Dgcr8. These cells are heavily depleted for microRNAs, allowing us to reintroduce specific microRNA duplexes and identify refined target sets. We used deep sequencing of small RNAs, mRNA expression profiling and bioinformatics analysis of microRNA seed matches in 3′ UTRs to identify target transcripts. Consequently, we have identified a network of microRNAs that converge on the regulation of several important cellular pathways. Additionally, our experiments have revealed a novel candidate for Dgcr8-independent microRNA genesis and highlighted the challenges currently facing miRNA annotation.Small RNAs such as microRNAs play important roles in embryonic stem cell maintenance and differentiation. A broad range of microRNAs is expressed in embryonic stem cells while only a fraction of their targets have been identified. We have performed large-scale identification of embryonic stem cell microRNA targets using a murine embryonic stem cell line deficient in the expression of  MicroRNAs (miRNAs) are important mediators of post-transcriptional gene regulation. They are RNA molecules ∼22 nt in length, responsible for guiding the RNA induced silencing complex (RISC) to mRNA molecules, predominantly through complementarity between the 5′ end of the miRNA (containing the seed region) and sequences within the 3′ UTR of the target molecules. This can lead to degradation of the targeted mRNA and inhibition of its translation. There are examples of these mechanisms acting independently, but it has recently become clear that in the majority of cases a miRNAs will reduce both protein and mRNA levels of a target Transcribed within much larger RNA sequences (pri-miRNA), miRNAs are released by series of RNase III processing reactions. In the nucleus the precursor miRNA (pre-miRNA) hairpin is released by the RNase III enzyme DROSHA, operating in conjunction with the double stranded RNA binding protein, DiGeorge syndrome critical region gene 8 (DGCR8) in vitro or in vivo conditions that may influence target selection, such as the co-expression of targets and miRNAs.The number of annotated miRNAs has expanded enormously over the course of the last decade. Currently there are 741 mouse-miRNA hairpins annotated in miRBase (release 18) in vitro model for development and a system that possesses considerable therapeutic potential. Recently, several systems have been developed that provide an insight into the roles that miRNAs play in ES cells by knocking out components of the miRNA processing pathway Dicer1 knockout cells maintain ES cell marker expression Dgcr8 knockout cells are able to revert to an undifferentiated state once the differentiation conditions are reversed Embryonic stem (ES) cells, which are derived from the inner cell mass of the blastocyst, are capable of self-renewal and are pluripotent, capable of differentiating into all somatic lineages. As such they provide an Dgcr8 and canonically processed miRNAs. This allows us to reintroduce miRNAs into a system with limited miRNA functional redundancy so targets should no longer be saturated by endogenous miRNA expression. Through a simple system by which miRNAs are reintroduced individually to these cells and subsequent mRNA expression changes are measured by microarray, we were able to partially rescue the wild-type ES cell mRNA expression profile and identify lists of mRNA transcripts that are likely targets of a number of miRNAs within wild type ES cells. In this way we are able to propose functions for individual miRNAs, uncover a broad network of the targets of miRNAs in ES cells and identify both basal transcription factors and the mediator complex as global/shared routes by which ES cell miRNAs appear to converge to regulate a wider cohort of secondary targets within these cells.We describe a mouse ES cell line depleted in the expression of Dgcr8 were disrupted. Targeted trapping of the second allele of Dgcr8 was performed in two independent gene trap cell lines from the BayGenomics resource   . This cont cells . This reDgcr8 depletion. In contrast, independent comparisons of the two homozygous mutant cell lines to their corresponding heterozygous control line identifies 2220 and 3101 transcripts with significantly altered expression, with approximately 73% of the smaller set of transcripts also found within the larger set. Thus, homozygous depletion of Dgcr8 results in a large number of significant expression changes at the mRNA level and those changes are highly consistent between replicates.To better understand the molecular consequences of miRNA depletion, mRNA expression was assayed for each of the cell lines. A comparison between WT and the individual heterozygous cell lines identified only 10 and 62 differentially expressed transcripts (in the two replicates respectively). This demonstrates that there is no broad effect on mRNA expression of heterozygous Dgcr8-depleted cells. Sylamer analysis of the list of transcripts ordered according to mRNA expression change (log fold change) upon Dgcr8 depletion  have been removed from miRBase  cells  cells . In all S) cells . In the S) cells .In order to judge the effectiveness of these experiments in generating significant miRNA target lists, calculations were performed to estimate the signal to noise ratio of each and an estimate was made of the number of targets in the target list above that which may have been expected by chance . The siggt1/tm1Dgcr8 cells should represent the reversal of the effect of the depletion of Dgcr8, at a molecular level. To confirm this, the expression profile of the Dgcr8-depleted cell lines when compared to the heterozygous controls was interrogated with each miRNA target list in turn, using Gene Set Enrichment Analysis (GSEA) Dgcr8 depletion. Hence, the regulation of transcripts within these gene lists is likely to be highly relevant in an ES cell context.The transfection of individual miRNAs into the Dgcr8 depletion. It is possible that the networks regulated by these miRNAs differ in complexity or they regulate a different number of in vivo targets. These enrichments may be more easily identifiable for miRNAs with large numbers of targets, a more significant effect on the down-regulation of those targets or which cause a broad depletion of an entire network, therefore having a more pronounced cumulative effect. On the other hand the seed enrichment for those miRNAs with fewer targets, whose role is to maintain homeostatic regulation of those targets under specific circumstances, or whose target networks may be corrected by feedback loops following perturbation may be harder to detect in this way. In the case of miR-298, it is not highly expressed in mouse ES cells so we were not expecting to identify a large number of in vivo targets that would be up-regulated upon miRNA depletion.There are several factors that may account for the differences in both the length of the target lists generated for each miRNA and the observed enrichments. The target lists of miR-302a and miR-106a are both the longest and most enriched amongst the genes up-regulated upon broad miRNA depletion in the ES cell system ). By contrast miR-292-5p and miR-298 have weaker seed sequence enrichments amongst significantly down-regulated genes following transfection and their proposed targets are not enriched among the genes up-regulated by It might be expected that ES cell expressed miRNAs regulate similar processes and may have considerable overlap between their target sets, leading to a robust layer of post-transcriptional regulation. It is expected that miRNAs with shared seed sequences will also share a considerable number of their targets due to the extent to which the recognition of miRNA target sites depends on the degree of complementarity between the target transcript and the miRNA seed region gt1/tm1Dgcr8 cells ) with both miR-291a-3p and miR-302a (7mer(2)), which may also suggest that they maintain functionally redundant roles through interaction with the same target sites. However, the transcripts bearing only the 7mer(3) seed sequence of miR-106a do not appear to be significantly affected by the transfection of miR-106a mimics into the m1 cells , so it im1 cells . In addim1 cells . Thus incell cycle network. Indeed, the targets of both miR-106a and miR-302a are significantly enriched within this category. Although the targets of miR-291a-3p are not enriched in this pathway, this is not surprising given the relatively small size of the miR-291a-3p target list. Our results therefore support a fundamental role for miRNAs in regulating the ES cell cycle.In order to better understand the function that each of these miRNAs play in mouse ES cells, enrichment analysis of annotation to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways Ccnd1, Cdkn1a and E2f1, all of which have been shown to targeted by the miR-17 family of miRNAs which includes miR-106a Cdkn1a, a key regulator of the G1/S phase transition, also appears in our miR-302a and miR-291a-3p target lists. These miRNAs share a seed sequence and as such this is consistent with the results of Wang et al.cell cycle pathway, Cdkn1c, a confirmed miR-25 target Our miR-106a target list includes cell cycle pathway. Considering the targets for those miRNAs either highly expressed in WT ES cells or for whom there is a clear seed sequence enrichment amongst those genes up-regulated following Dgcr8 depletion  and E2f2  both appear to be potentially targeted by three miRNAs, while Skp2 (miR-21 and miR-302a) and Cyclin D1 (miR-106a and miR-302a) may be targeted by two each. Further to this, miR-302a appears to target multiple members of the anaphase promoting complex. This implies a degree of cross-regulation and redundancy not only between miRNAs with shared seed sequences (miR-291a-3p and miR-302a) but also both amongst miRNAs with no shared seed identity and through the regulation of multiple components of a functional complex.It is intriguing to note that multiple miRNAs appear to converge on a number of genes in the epletion , Cdkn1a While miR-302a and miR-106a appear to make the greatest contribution to regulation of the cell cycle, the targets of miR-21 are also significantly enriched in this pathway despite possessing a completely different seed sequence. The connection of miR-21 to cell cycle regulation is perhaps expected as this miRNA is known to have altered expression in a number of cancers. It is clear from these interactions and the additional associations demonstrated in this study, that a complex network of miRNAs regulate the progression of the ES cell cycle.basal transcription factors KEGG Pathway . To validate the clones, nested RT-PCR reactions were performed with primers in the gene trap cassette sequence and exon sequences upstream of the insertion site . PCR products were subsequently purified and sequence confirmed. Each of the cell lines was subcloned and reconfirmed by RT-PCR.Two independently derived cell lines were acquired from BayGenomics (XG058 and XH157), each with a gene trap in a single allele of NotI digested BAC clone bMQ-62C21 AscI restriction site . The cloning strategy for the Dgcr8 insertion-type targeting construct is shown in AscI fragment contains exons 4–8 of Dgcr8 and was cloned into pR3R4_AsiSI, replacing the chloramphenicol resistance gene and ccdB cassette. The fragment was then transferred to the pL3/L4_(+)_GT1T2hygroP2EGFP gene trap plasmid through an in vitro L/R clonase reaction (Gateway L/R clonase II (Invitrogen)). Prior to electroporation, the plasmid was linearised in the homology region at a unique HindIII site in intron 6 of Dgcr8 (ENSMUST00000115633). Hygromycin resistant clones were picked and expanded. Nested RT-PCR between Dgcr8 specific primers that bind to exons upstream of both the gene trap insertion sites and homologous region and primers that are common to both gene trap cassettes were used to detect the trapped and targeted alleles . Clones that express both the BayGenomics gene trap and the targeted gene trap alleles were considered to be homozygous mutant lines. Products representative of each of the expected amplicon sizes were confirmed by sequencing.2 in gelatinised tissue culture treated plates and flasks and electroporated as described +/+Dgcr8 (E14) gt1/+Dgcr8 and gt2/+Dgcr8 cells were maintained in media supplemented with 150 µg (active)/ml G418 (Geneticin - Gibco). Following the electroporation of the gene-targeting construct, cells were selected at 120 µg/ml Hygromycin B  and maintained at 100 µg/ml Hygromycin B. To assay for β-galactosidase activity, cells were washed with PBS, fixed in 0.2% gluteraldehyde and stained with 1 mg/ml Xgal as described Cell lines were maintained feeder-free in ES cell medium (GMEM+10% serum+LIF) at 37°C, 7% COCells were maintained for 4 days in non-selective media prior to lysis. RNA was purified by Trizol and RNeasy according to the manufacturers protocol. RNA was quantitated on an Agilent Technologies 2100 Bioanalyser using a Eukaryotic Total RNA Nano Chip. Polyadenylated RNA was purified with the PolyATract mRNA Isolation System III (Promega) and quantitated as above to ensure removal of rRNA. Sample concentrations were equalized, and separated by denaturing agarose gel electrophoresis in the presence of SybrGreen (Invitrogen) and a 0.5–10 kb RNA ladder (Invitrogen). Separated RNA was transferred overnight by capillary blot to Hybond XL membrane  and UV cross-linked (UVP).+/+Dgcr8 Trizol-purified-RNA derived cDNA followed by subcloning and reamplification. Probes were amplified with the KOD Hot-Start PCR kit (Novagen) using primers GCTGGGCTGTTGTCTCCATA and CATCTTGGGTTTCTTCCGAGT (3′) or CGACGACCCATTCAACTTCT and TCGAGCACTGCATACTCCAC (5′) and purified by Qiagen Qiaquick Gel Extraction. The probe fragments were A-tailed , AmpliTaq (Perkin Elmer), ∼250 ng DNA), and cloned using pGEM-T-Easy (Promega) with the Roche Rapid Ligation Kit and MACH1 cells (Invitrogen). Transformant colonies were confirmed by colony PCR with flanking plasmid primers GTAAAACGACGGCCAGT and GGAAACAGCTATGACCATG. Plasmids were prepared using a Qiaprep Spin Miniprep Kit (Qiagen) and inserts were confirmed by sequencing. The probes were finally amplified by KOD Hot-Start PCR and the probe specific primers and purified by Qiaquick Gel Extraction (Qiagen).RNA probes were prepared by amplification from Amplified probes were radiolabeled using the Random Labeling kit (Invitrogen). Hybridisations were performed overnight in PerfectHybTMPlus buffer (Sigma) at 55°C. The membrane was washed to 0.1× SSC with 0.1% SDS at 55°C and imaged by phosphoimager  for 2.5 hours.Cells were maintained for 4 days in non-selective media prior to protein purification and lysed while subconfluent. The cells were lysed in protein lysis buffer ) by repeated passage through a 21 G hypodermic needle. Samples were quantified with Bradford Reagent. 50 µg of each reduced sample was size separated on a 4–12% Bis-Tris Gel (Invitrogen). The proteins were transferred to a Hybond-ECL filter. Oct4 and α-tubulin were detected using primary antibodies  and peroxidase conjugated secondary antibodies (Sigma-Aldrich A4174 and A6782) were used to visualise each protein sequentially with Western Lightning reagents.gt1/tm1Dgcr8 cells were cultured for 4 days in non-selective media and plated to gelatinised 6 well plates at 96×104 cells per well. Cells in each well were transfected after 3 hours. 240 pmoles of miRNA mimic were added to 240 µl OptiMEM I (Gibco). 7.2 µl of Lipofectamine 2000 (Invitrogen) was added to further 240 µl of OptiMEM and incubated for 5 minutes at room temperature. Both solutions were mixed gently and then combined. This mixture was incubated for a further 25 minutes at room temperature. The media on the cells was replaced with 2.4 ml of fresh, non-selective media. The miRNA-lipid complexes in the OptiMEM mixture were then transferred to this media and the wells were mixed gently. 5 hours later, the media was aspirated from the cells and replaced with a further 7.2 ml of non-selective media. 10 hours after the initiation of transfection the cells were lysed for RNA with 1 ml of Trizol (Invitrogen). Duplicate miRNA transfections were performed and expression profiles were compared to those of control mimic transfections prepared in parallel.gt1/tm1Dgcr8 cells. Both of these miRNAs are highly expressed in the wild type mouse ES cells, are down-regulated in the gt1/tm1Dgcr8 and gt2/tm1Dgcr8 cell lines and their seeds are enriched in the 3′ UTRs of transcripts up-regulated upon Dgcr8 depletion. The Sylamer algorithm was used to analyse gene lists ordered according to differential expression between the miRNA transfected cells and those transfected with a control duplex. This identified 10 hours as the time point at which the seed sequences for each miRNA were most significantly enriched amongst the down-regulated genes.To determine the optimal time post-transfection at which to lyse cells to identify the gene set most enriched for primary miRNA effects, transfected cells were lysed in a time-series post-transfection and their mRNA expression was profiled at each time point. Initially, miR-291a-3p and miR-25 were transfected into the miRIDIAN Negative Control #2 (Dharmacon CN-002000-01-05)miRIDIAN mmu-miR-291-3p mimic (Dharmacon C-310470-01-0005)miRIDIAN mmu-miR-25 mimic (Dharmacon C-310564-01-0005)miRIDIAN mmu-miR-302 mimic (Dharmacon C-310483-05-0005)miRIDIAN mmu-miR-292-5p mimic (Dharmacon C-310471-03-0005)miRIDIAN mmu-miR-106a mimic (Dharmacon C-310488-07-0005)miRIDIAN mmu-miR-21 mimic (Dharmacon C-310515-05-0005)miRIDIAN mmu-miR-298 mimic (Dharmacon C-310479-07-0005)Trizol purified RNA was cleaned up with an RNeasy MiniElute Cleanup Kit (Qiagen). 500 ng of RNA was amplified and labeled with the Illumina Total Prep RNA Amplification Kit (Ambion). Microarrays were processed by the Sanger Institute Microarray Facility. Briefly, 1500 ng of biotinylated cRNA was hybridised to Illumina expression BeadChips . Bead chips were processed following the manufacturer's instructions and analysed using BeadStudio software v.3.1.8 or GenomeStudio version 1.1.1 (Illumina). All array data has been submitted to ArrayExpress (E-MTAB-418 and E-MTAB-968).http://www.bioconductor.org/) with additional packages . Samples derived from each array version were combined into 2 independent sets, variance-stabilising transformation (VST) transformed and quantile normalised lumi package of Bioconductor. Probes from both the v1.1 and v2 formats were mapped to Ensembl gene annotation v56 . Additional annotation for these transcripts, including Gene ID, Entrez ID, Gene symbol and Description were also derived from Ensembl. When no Ensembl transcript could be found for a probe, a RefSeq transcript was assigned if available from Illumina chip annotation along with additional annotation (MouseWG-6_V1_1_R4 and MouseWG-6_V2_0_R2). Probes with no transcript annotation were removed form the set used for transcriptional analysis. Where multiple probes mapped to the same target transcript, the probe with the highest inter-quartile range across arrays was retained. The remaining probes were considered for differential expression between samples. Samples ascribed to different array versions and arrays relating to miRNA transfection and broad cell line profiling experiments were analysed independently at this stage.Analysis of the expression array data was conducted in R/Bioconductor  cutoff of 0.1 were used where required.Differential gene-expression analysis was carried out on these data by performing an empirical bayes t-test with Benjamini Hochberg multiple-testing correction via the Transcripts were considered as possible miRNA targets if they were significantly down-regulated at least 1.2×  in the presence of a transfected miRNA mimic relative to the expression in cells transfected with a control duplex. Since the v2 arrays appear to be more sensitive, a p-value cutoff of 0.05 was used for these miRNA transfection experiments, compared to 0.10 for the v1.1 arrays. Annotation information was assigned to the significant probes according to Ensembl v56.For the purposes of this study 7mer(1A), 7mer(2) and 7mer(3) and 8mer(1A) seed sequences were defined as follows:7mer(1A): A 6 nucleotide sequence complementary to positions 2 to 7 of the miRNA followed by an adenosine.7mer(2): A 7 nucleotide sequence complementary to positions 2 to 8 of the miRNA8mer(1A): A combination of the two seed sequences above.7mer(3): A 7 nucleotide sequence complementary to positions 3 to 9 of the miRNASylamer was used to count the number of corresponding miRNA seed sequences within the unmasked 3′ UTRs of annotated target transcripts. Transcripts without one or more corresponding 7mer(2) or 7mer(1A) seed sequence were removed from the target lists.Dgcr8 depletion. In the comparisons between each miRNA transfection and the corresponding control, the targets of the miRNA should be relatively down-regulated by the miRNA transfection. Heterozygote and homozygous mutant samples derived from either the gt1/+Dgcr8 and gt2/+Dgcr8 lineages were considered as replicates for the comparison of expression between Dgcr8-depleted cells and heterozygotes and heterozygote and WT cells.Sylamer was used to verify miRNA-directed changes of gene expression. The expectation is that targets of many miRNAs will be up-regulated upon Briefly, Sylamer tests for miRNA effects by searching sorted lists of genes for enrichment or depletion of all possible words complementary to the seed regions of miRNAs. The method uses the hypergeometric statistic, comparing the counts of each word in all the 3′ UTR sequences, before a cutoff in a sorted gene list, to the counts in the remaining genes after that cutoff. Gene lists were produced by sorting the transcripts with annotated 3′ UTR sequence , according to the observed differential expression between different samples. Sylamer parameters used were -k 7 (words of length 7), -grow 500 , -m 4 (use Markov correction for nucleotide biases using observed frequencies of words of length 4) and -words word_file (only test words present in ‘word_file’). The words considered were all the 7 nt words complementary to positions 1–8 of annotated mouse miRNAs for which at least 10 reads were sequenced in any of our Illumina GA experiments, always using an adenine as the base matching the first position of the miRNA As a measure of the specificity of this target identification method a signal to background ratio was determined for each miRNA target list. The proportion of transcripts that were down-regulated significantly upon the introduction of the miRNA, which possess a miRNA seed in their 3′ UTR (7mer(2) or 7mer(1A)) was divided by the proportion of transcripts whose expression was altered less than 1.1 fold in the same experiment (control set) and which possess the same seeds in their UTRs. Similarly the sensitivity of each target list was calculated as the number of significantly down-regulated transcripts that possess a seed sequence for the specific miRNA beyond the number of transcripts that would be expected given the proportion of transcripts with seeds in the control set.org.Mm.eg.db) were removed from the corresponding analyses. After filtering, the genes belonging to each miRNA target list were compared to the rest of the genes from the corresponding array using the GOstats package to test for enriched KEGG pathways. A p-value significance cutoff of 0.01 was used.Probes used in the initial expression analyses were mapped to Entrez IDs (based on Ensembl v56 annotation or Illumina annotation files (see above)) and used as a Gene Universe. For this analysis probes without Entrez Gene ID annotation were removed in addition to duplicate IDs. Furthermore, Entrez IDs without any associated KEGG annotation . The gene subsets tested for enrichment consisted of the miRNA target lists derived from the transfection of the miRNA mimics (see above). Where target lists were derived from experiments performed on an array version that differed from that used in the comparison of expression in heterozygous cell lines to the Dgcr8-depleted cells, only transcripts whose expression was interrogated by both array versions were retained for this analysis. The number of permutations used in the GSEA analysis was 40000, which allows for the estimation of a minimum p-value detection of 2.5E-5.Gene Set Enrichment Analysis (GSEA) was performed using the “GSEAPreranked” method The molecular interaction network was retrieved from Pathway Commons (Feb 2011) et al. Supplementary org.Mm.eg.db library in R/Bioconductor. In the case of multiple Entrez IDs the first was selected. All NAs and duplicate IDs were removed from the gene sets.For each miRNA, targets without an annotated Entrez ID were removed from the lists along with duplicate Entrez IDs. The miR-294 and let-7a target sets were derived from Melton The initial interaction network consisted of 4808 nodes with 55894 edges, corresponding to an average node degree of 23.3. In this network, there were many nodes of very high degree, namely two nodes of degree > = 400, 4 nodes of degree > = 300, 28 nodes of degree > = 200, and 218 nodes of degree > = 100. Such ‘hub’ nodes obscure cluster structure, as they tend to pull together many nodes into coarse clusters.This network was reduced using a k-nearest neighbour approach, with k chosen as 100. With this approach, an edge E with weight w between two nodes a and b is kept only if w is in the top k edge weights for 1) the edges emanating from a and 2) the edges emanating from b. The input network could not be submitted to k-NN (k-nearest neighbour) reduction straight away, as it is a simple network with all edge weights equal to 1. The chosen approach was to add to each edge weight a number proportional to the number of triangles in which the edge participates. This preprocessing step, promoting edges in accordance with the number of secondary connections, allowed the application of k-NN reduction. Following this reduction with k = 100, the resulting network has 46788 edges, corresponding to an average node degree of 19.5. In the resulting network the highest node degree is now 100.The reduced network  was submitted to MCL clustering. Two clusterings were considered, at different levels of granularity, with the first a superclustering of the second. The first, coarse clustering was obtained by using inflation 1.3, the second was obtained by subclustering the k100 network restricted to the first clustering, with inflation set to 2.0. The clusters were annotated by running a GO-enrichment script written in R, using the customary hypergeometric test. All GO categories  were considered. The second clustering was deemed more informative, as large clusters split into smaller units with more specific annotation. For example, the largest supercluster with 433 nodes and annotations ‘ribonucleoprotein complex (8.7e-71), ‘RNA splicing’ (1.1e-47) and ‘RNA processing’ (2.5e-47) split into a cluster with 92 nodes , a cluster with 85 nodes , a cluster with 85 nodes , a cluster with 82 elements , a cluster with 79 elements  and several smaller clusters. When considering the fifty largest clusters in the second clustering, the median of the best P-value associated with each of the clusters (referring to a GO-term enriched for such a cluster) is 6.811652e-10. For each cluster, the two best scoring GO-terms were used to annotate the cluster in the heatmap . For praThe heatmap shows for each list of target genes (corresponding to a list of nodes in the network) and for each cluster the support for that list in that cluster. This number (the support) is the sum of the support of each individual node (from the list) for that cluster, normalised by cluster size. The support of a node is the sum of edge weights (in the k100 network) for its edges that connect it to (other nodes in) the cluster, divided by the total sum of weights of all its outgoing edges.It is thus possible for a cluster to give support to a node that is not part of the cluster, simply by virtue of the node having neighbours in that clusters. Additionally, the heatmap shows numbers in the cells. Such a number indicates, for the cluster and target list specific to that cell, the number of genes shared between the cluster and the list.Cells were maintained for 4 days in non-selective media prior to lysis. RNA was purified with Trizol (Invitrogen). Large RNA was removed from the small RNA fraction using the RNeasy Mini Kit (Qiagen), and small RNAs were collected by isopropanol precipitation and quantitated on an Agilent Technologies 2100. Sequencing libraries were prepared by an adapted version of Illumina “Preparing Samples for Analysis of Small RNA” protocol version 1 (2007). Initially, the 3′ adaptor was added to heat denatured small RNA fraction and ligated with RNA ligase in the presence of 20% DMSO and RNase Out (Invitrogen). The RNA between 35–65 bp was size separated on a Novex 15% TBE-urea gel, eluted and ethanol precipitated in the presence of GlycoBlue (Ambion). Heat denatured RNA was ligated to the 5′ Adapter as before. RNA of 60–100 bp in size was purified by 10% TBE-urea gel electrophoresis and recovered as above. The RNA was reverse transcribed and the cDNA was amplified by PCR using the smRNA primer 2 and RT primer (Illumina) with Phusion Taq (NEB). Heat denatured cDNA was purified by 10% SequaGel PAGE gel electrophoresis and recovered in 0.3 M NaCl followed by ethanol precipitation and quantified by Agilent Technologies 2100 Bioanalyzer. The Illumina libraries were Solexa sequenced by the Sanger Institute Core Sequencing Facility . Sequencing data has been submitted to ArrayExpress and ENA (E-MTAB-975).FASTQ files from each sequencing lane were processed by R/Bioconductor using the ShortRead and Biostrings packages. After verifying that the sequencing quality was acceptable for all libraries, the 3′ adapter sequence was trimmed from all the reads with the trimLRPatterns function. The distribution of read lengths confirmed the expected enrichment of miRNAs (a peak of ∼22 nucleotides) for the wild type and heterozygous samples, while this was essentially absent in the Dgcr8-depleted samples. Reads were then trimmed if they contained runs of ambiguous nucleotides (three Ns in a window of five bases) of if they contained any number of Ns within 2 bases from the end of the read. A low-complexity filter was implemented to remove all reads that consisted mostly of runs of identical nucleotides, di-nucleotides or tri-nucleotides. The remaining sequences of lengths between 16–30 were kept for further analysis, collapsing them into unique sequences and keeping track of the observed depth of sequencing.The A dataset of all mouse non-coding RNA sequences was obtained by extracting all the hairpin sequences from miRBase v14 and all RNA genes and predictions from Ensembl v56. The distinct sequence reads were mapped against this dataset using SSAHA2 The normalisation strategy consisted of using the total depth of reads mapping to ncRNAs (excluding the miRNA and mixed categories) and scaling all the libraries to have an equivalent depth to the WT sample .Figure S1Schematic illustration of the gene targeting strategy used in this study.A) Map of vectors introduced in this study. The pR3R4AsiSI plasmid is a Gateway shuttle vector for cloning genomic DNA fragments. Positive and negative selection cassettes, CmR and ccdB, respectively are flanked by AscI restriction sites and attR3 and attR4 Gateway sites. The pL3L4_(+)_GT1T2hygP2EGFP plasmid contains a gene trapping cassette and attL3 and attL4 Gateway cloning sites to allow transfer of cloned genomic DNA fragments. The gene trap cassette is composed of the En2 splice acceptor, hygromycin resistance gene (HygroR), Enhanced Green Florescent protein gene (EGFP) and the SV40 polyadenylation site (SV40 polyA). The T2 and P2 sites cause ribosome skipping and are included for optimal expression of the resistance marker and fluorescent reporter B) Cloning of the homology region into the pL3L4_(+)_GT1T2hygP2EGFP vector. See C) Schematic of the Dgcr8-targeted trapped and gene trapped alleles. The pGT1T2hygP2EGFP_Dgcr8 plasmid is an insertion-type gene-targeting vector containing exons 4 to 8 of Dgcr8. The vector is linearised within the homology region at a unique HindIII site prior to electroporation. The resulting targeted events cause a duplication of the homology region, placing the hygromycin-EGFP cassette downstream of exon 8. The BayGenomics gene trap cassette contains a β-geo reporter cassette, conferring G418 resistance and β-galactosidase activity, inserted downstream of exon 9. Insertion of the targeted cassette into the Bay Genomics gene trap allele will silence the β-geo reporter (reporter .(TIFF)Click here for additional data file.Figure S2Xgal staining of cell lines to determine β-geo  activity associated with the initial gene trap. Xgal-staining confirmed that in the selected heterozygous cell lines , the insertion of the second gene trap had disrupted the expression of the fusion transcript containing the original downstream construct and silenced the β-galactosidase activity of the fusion protein produced. This staining demonstrated that both gene traps are inserted within the same allele of the target gene. In contrast the homozygous mutant cell lines retained positive x-gal staining confirming that the gene traps must be within separate alleles of the gene. The inserted pane shows the nuclear localisation of the β-galactosidase activity of the β-geo fusion protein.(TIFF)Click here for additional data file.Figure S3RNA blot of Dgcr8 derived transcripts assessing the expression of wild type and fusion transcripts. RNA derived from the two heterozygous cell lines were separated  alongside the 2 homozygous mutants (gt1/tm1Dgcr8 and gt2/tm1Dgcr8) and the wild type cell line (Dgcr8+/+). Additionally, RNA samples from the parental gene trap cell lines were also blotted (gt1/+Dgcr8 and gt2/+Dgcr8). The blot was hybridised sequentially with radiolabelled probes that either anneal to the 5′ (top) or 3′ (bottom) ends of the Dgcr8 transcripts. The expected transcript sizes are shown to the right of the blot. Size estimates for the gene-trapped transcripts are based on ENSMUST00000115633 and include the gene trap cassettes up to the polyadenylation sites. The nature of the smaller wild type transcript is unclear although the second small wild type transcript has previously been observed in mouse (TIFF)Click here for additional data file.Figure S4Read counts for each class of ncRNA in each cell line.A) Raw small RNA mapped read counts. B) Equivalent read counts after scaling to the non-miRNA non-coding RNA population in the WT sample.(TIFF)Click here for additional data file.Figure S5Total read counts for each RNA species compared between cell lines pre-normalisation.(TIFF)Click here for additional data file.Figure S6Western blot comparing the expression of Oct4 in Dgcr8-depleted, heterozygous and wild type cell lines. The same blot was treated with antibodies for both Oct4 and α-tubulin (loading control).(TIFF)Click here for additional data file.Figure S7Global expression profiles to determine miRNA-dependent transcriptional effects.A: Sylamer plots comparing the expression profiles of Dgcr8gt1/tm1 cells transfected with a miRNA mimic  and a cel-miR-239b control miRNA. For a full description see B: GSEA enrichment plots Dgcr8 in homozygous mutant cell lines. For a full description see (TIFF)Click here for additional data file.Figure S8Overlap in target transcripts between each of the miRNAs. The percentage overlap represents the proportion of the transcripts within the smallest target set which overlap the larger set in each pairwise comparison between miRNA target sets. Bonferroni corrected hypergeometric P-values were calculated for each overlap. The universe consisted of those transcripts interrogated in the differential expression analyses. The number of potential targets identified for each miRNA is recorded beneath the miRNA name. In cases where targets were derived from alternative microarray versions, only transcripts interrogated by both platforms were considered in the comparison.(TIFF)Click here for additional data file.Figure S9Assessing the potential for functional redundancy between miR-106a and miR-302a through a shifted 7mer(3) seed. A cumulative graph is presented of the relative effect of each miRNA seed sequence (7mer (1A), 7mer (2) and 7mer (3)) on transcripts which contain at least one seed from the relevant category and for which the seed is not part of a longer seed matching site (achieved through the exclusion of transcripts containing adjacent 7mers)  when compared to a 1/10 sampling of all transcripts represented on the array (Black), following the transfection of miR-302a or miR-106a miRNA mimics. P-values displayed were calculated using a Wilcoxon or T-test to determine if the relative distribution of the seed bearing transcripts, according to log fold change (logFC), differ significantly from the bulk of the other transcripts following miRNA transfection. The x-axis represents the relative logFC following miRNA transfection when compared to the cel-miR-239b. The y-axis is the cumulative percentage of each target set or transcripts represented on the array.(TIFF)Click here for additional data file.Figure S10Investigation of the seed sequences found in miR-106a and miR-302a targets.A) A Venn diagram of transcripts within the target lists of both miR-106a and miR-302a. Shown are the numbers of transcripts found exclusively within the miR-302a target list and the miR-106a target list in addition to those shared by both. B) The class of seed sequence found in the 3′ UTRs of target transcripts. The nature of the miR-106a and miR-302a seed sequences found within the 3′ UTRs of the transcripts found exclusively in the miR-106a target list, the miR-302a target list or those transcripts within both target lists. The extended 8mer refers to the target sequence AGCACTTT, which is complementary to the accepted seed sequences of both miR-302a and miR-106a. The miR-302a and miR-106a target sites exclusively refer to those target sites that do not overlap these extended sites and are therefore not expected to be targeted by both miRNAs.(TIFF)Click here for additional data file.Figure S11miRNAs found to target transcripts of proteins found within cluster 12 of the interaction network. Hexagons represent proteins and grey lines represent known interactions. Stars mark those genes in the sub-network targeted by miRNAs either in this study or in the work of Melton et al.(TIFF)Click here for additional data file.Figure S12Relative disruption of miRNA targets within mediator associated cluster. Log fold change of transcripts associated with the miRNA targets from within cluster 12  upon the addition of each miRNA relative to the control miRNA (Left) and the log fold change upon the depletion of all miRNAs (Right). Stars represent those transcripts selected as potential targets of the transfected miRNA.(TIFF)Click here for additional data file.Table S1Transcripts identified as probable miRNA targets. Transcripts identified as the probable targets of each miRNA over-expressed within this study. Each table includes the number of relevant seed sequences within the transcript's 3′ UTR, the log fold change and associated p-value of each transcript following transfection of the miRNA when compared to a cel-miR-239b control miRNA Click here for additional data file.Table S2Estimates of the signal to noise ratio of each of the target lists produced as part of this study.(XLSX)Click here for additional data file.Table S3KEGG Pathway analysis results. Significant KEGG pathway terms over-represented amongst the targets of the miRNAs with a significance cutoff of 0.01 in each case.(XLSX)Click here for additional data file.Table S4Sequences mapping to RNA class sets. A summary of the number of sequences mapping to each RNA class set. Columns in bold italics represent those RNA classes used to normalise the samples.(XLSX)Click here for additional data file.


We also observed that the expression of VEGF increased significantly during the development of hepatic fibrosis and CCl4 was found to induce hepatocytes to secrete VEGF, which led to the activation and proliferation of HSCs. Bevacizumab was also found to block the effects of the hepatocytes on the activation and proliferation of HSCs. Our results suggest that bevacizumab might alleviate liver fibrosis by blocking the effect of VEGF on HSCs. Bevacizumab might be suitable as a potential agent for hepatic fibrosis therapy.Angiogenesis is a fundamental part of the response to tissue injury, which is involved in the development of hepatic fibrosis. Vascular endothelial growth factor plays an important role in angiogenesis. The expression of VEGF is increased during hepatic fibrogenesis and correlates with the micro-vessel density. In this study, we investigated the effects of bevacizumab, an anti-angiogenetic drug, on the formation of hepatic fibrosis. We found that bevacizumab could attenuate the development of hepatic fibrosis and contribute to the protection of liver function. Bevacizumab was also found to downregulate the expression α-SMA and TGF-β1, which have been reported to be profibrogenic genes  Hepatic fibrosis is characterized by excess production and deposition of extracellular matrix (ECM), which leads to loss of liver function and structure disruption of liver tissue4) intoxicationHepatic stellate cells (HSCs) play an important role in the development of hepatic fibrosis. HSCs are considered as a key target in anti-fibrotic therapy because of their role in ECM accumulationBevacizumab, a full-length humanized monoclonal antibody, is a therapeutic candidate suitable for use as a direct inhibitor of angiogenesis. Its antiangiogenic efficacy is attributable to its ability to bind and neutralize all isoforms of VEGF-A in vivo. We then, examined the role of bevacizumab in the proliferation and activation of HSCs in vitro. Our results demonstrated that bevacizumab administration could alleviate liver fibrosis by inhibiting activation and proliferation of HSCs.In this study, we investigated the effects of bevacizumab on liver fibrosis. Carbon tetrachloride was used to establish a hepatic fibrosis animal model suitable for observation of the effect of bevacizumab 4-induced hepatic fibrosis. As shown in 4 in rats. Semiquantitative analysis of the ECM area revealed that bevacizumab significantly reduced the area of ECM (Sirius red staining) after injection in the CCl4-treated fibrotic livers (We assessed the effects of bevacizumab in a rat model of CClic livers. In CCl4 P<0.05) .As shown in 4. Bevacizumab injection was found to remarkably down-regulate the expression of the genes related to liver fibrosis relative to the control groups  were used to detect the expression of α-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1) in the liver tissues. These have been reported to be important for the development of hepatic fibrosis. The results demonstrated that expression of α-SMA and TGF-β1 was very low in normal liver tissue. However, a significant increase in gene expression was observed with the development of hepatic fibrosis induced by CCll groups .We also examined the expression of VEGF in liver tissue and serum. As shown in 4. As shown in The expression of VEGF was higher in fibrotic hepatic tissue than in healthy tissue. Real-time PCR and ELISA were used to examine VEGF expression in hepatocytes after exposure to CCl4. We then detected the gene expression of fibrotic markers in HSCs treated with the conditioned medium. As shown in We have demonstrated that the expression of VEGF increased significantly during the formation of hepatic fibrosis and that bevacizumab could effectively attenuate hepatic fibrosis. HSCs have been shown to play a central role in the development of hepatic fibrosis. HSCs are considered a key target in anti-fibrotic therapy because of their role in ECM accumulation. The conditioned medium was collected from hepatocytes which were treated with CCl4. We found that the up-regulation of α-SMA and TGF-β1 in conditioned medium-treated HSCs was cancelled by bevacizumab . A rat normal liver cell line-BRL and an activated HSCs cell line-HSC-T6 were obtained from the cell bank of the Chinese Academy of Sciences .Male Sprague-Dawley rats (190±15 g) were housed under standard animal laboratory conditions in the specific-pathogen-free-grade animal room at the Experimental Animal Center of the Second Military Medical University. The rats had free access to standard rat chow and water. This study was approved by the Local Ethical Committee of the Second Military Medical University.4 at a dose of 2.4 ml/kg twice per week for 8 weeks2 exposure and liver tissues were harvested.The hepatic fibrosis model of SD rats was induced by subcutaneous injection of 40% CClAll paraffin-embedded liver tissues were HE stained for histopathological examination. Sirius red staining and Masson's trichrome staining were used to assess collagen levels. The red-stained areas in the Sirius Red stained sections were assessed with an image analyzer  for semiquantitative analysis. The percentage of the Sirius Red was used to demonstrate the differences in each groups. Immunohistochemical examinations were used to detect the expression of α-SMA , and TGF-β1 .Total hepatic hydoxyproline levels were determined in the hydrolysates of liver samples as described previously Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin(TB)were assessed by the kits from Sigma-Aldrich.2 in a humidified incubator.Primary HSCs were freshly isolated as described previously 4(5 mmol/L) for 12 hours. Then the culture medium was replaced with fresh DMEM. After another 24 hours of culture, the conditioned medium was obtained by collection and 0.22 µm filtration of the supernatant medium from BRL cells.BRL cells were stimulated with CClELISA assays were performed using a commercial VEGF ELISA kit . Samples were diluted 10-fold in deionized water before the assay. Assays were performed in duplicate, and readings were compared using standard curves obtained with standard protein provided with the kit. Samples were collected in triplicate, and means and standard deviations were compared using the t-test.3 cells/well) were seeded in 96-well plates for overnight, we changed the medium with conditioned medium and continued to culture these cells for 24 hours. When the treatment was completed, 10 µl solution of Cell Counting Kit-8 was added in each well. The plate was continuously incubated for 2 hours. Finally, the absorbance of sample taken from each well was measured by microplate reader  at 450 nm.The measurement of viable cell mass was assessed by Cell Counting Kit-8 . Cells (5×103 cells/well for overnight. Then we changed the culture medium with conditioned medium and continued to culture these cells for 24 hours. Twenty microlitres of MTT (5 mg/ml) were added to each well and the plate was continuously incubated for 4 hours. The formazan crystals were dissolved in 200 µl of DMSO. Finally, the absorbance of sample taken from each well was measured by microplate reader  at 490 nm.In order to measure the effects of conditioned medium on the viability of HSC-T6 cells, the cells were seeded in 96-well plates at a density of 5×10Total RNA was extracted from the cells using Trizol reagent . The cDNA was synthesized using MMLV reverse transcriptase  and 2 µg total RNA and oligo dT18-primers. Real-time PCR was performed in triplicate using a SYBR PrimeScript RT-PCR Kit . Total RNA was normalized by endogenous β-actin mRNA. The level of mRNA expression is presented as fold change relative to an untreated control. The primer sequences used in realtime-PCR are shown in P<0.05 was considered to be statistically significant.Data sets were analyzed by analysis of variance (ANOVA) with a posteriori contrast by least significant difference for comparisons among multiple groups and by Student t-test for comparison between two groups using the Microsoft Excel Analysis Tool Pak . The data, collected from at least three separate experiments, was showed as mean ±SEM.


The mechanism may be associated with the reduced expression of cancer stem cell markers HIF-2α, Oct-4 and ABCG2. 5-Fu and celecoxib have a synergistic antitumor effect. The mechanism associated with the amelioration of resistance to chemotherapy in gastric cancer and the enhancement of the effect of chemotherapy may be via the reduction of the expression of HIF-2α, ABCG2, Oct-4 and other cancer stem cell markers in the tumor tissues.5-Fluorouracil (5-Fu) is one of the most commonly used drugs to treat gastric cancer; however, drug-resistance in cancer cells reduces the efficacy of 5-Fu. Celecoxib may be able to reduce resistance to 5-Fu chemotherapy. The aim of the present study was to investigate the inhibitory effects of a combination of 5-Fu and celecoxib on implanted gastric cancer xenografts in nude mice and to elucidate the underlying mechanism. A tumor-bearing nude mice model was established. The mice were divided into blank control, 5-Fu, celecoxib and combination groups. The weight change and the tumor inhibition rate in each group were calculated. Immunocytochemistry, reverse transcription-polymerase chain reaction and western blotting methods were used to observe hypoxia-inducible factor-2α (HIF-2α), ATP-binding cassette transporter G2 (ABCG2) and octamer-binding transcription factor 4 (Oct-4) expression in the SGC7901 cells. Inhibition of the growth of the implanted gastric cancer was observed in the 5-Fu, celecoxib and combination groups. In the celecoxib and combination treatment groups, the mean tumor mass was significantly less than that in the control group (P<0.05), and the mean tumor mass in the combination treatment group was significantly less than that in the 5-Fu group (P<0.05). The tumor inhibition rates in the 5-Fu, celecoxib and combination groups were 26.36, 59.70 and 88.37%, respectively. The combination group exhibited the highest inhibition rate; the inhibition rates of the combination and celecoxib groups were significantly higher compared with the 5-Fu group (P<0.05). The expression levels of HIF-2, ABCG2 and Oct-4 mRNA and protein were high in the blank control group, and were further increased in the 5-Fu group. However, in the celecoxib and combination groups, the expression levels were lower compared with those in the control group. Significant differences were identified among the 5-Fu, celecoxib and combination groups (P<0.01). Celecoxib has antitumor effects  On the basis of these previous experiments, the current study used human gastric cancer cells transplanted in nude mice to investigate the inhibitory effects of celecoxib on SGC7901 cell growth in vivo. Whether the combination of 5-Fu and celecoxib is able to reduce the expression of stem cell markers HIF-2α, ABCG2 and Oct-4 in human gastric carcinoma tumors transplanted into nude mice and improve the resistance to 5-Fu chemotherapy was also examined.Chemotherapy is one of the primary treatments for gastric cancer . AlthougThe human gastric cancer cell line SGC7901 was given as a gift from Professor Yuguang Feng of the Affiliated Hospital of Weifang Medical College . 5-Fu was acquired from Jiangsu Zhenguo Pharmaceutical Co., Ltd. . Celecoxib was purchased from Pfizer . Rabbit anti-HIF-2α, Oct-4 and ABCG2 polyclonal antibodies were from Abcam . Immunohistochemistry kits (SP-9000) were purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd. . TRIzol reagent was from Invitrogen Life Technologies, and the reverse transcription (RT) and polymerase chain reaction (PCR) kits were purchased from Fermentas (Thermo Fisher Scientific). The protein extraction kit was from Biyuntian Co. . The western blot enhanced chemiluminescence kit was from Thermo Fisher Scientific. The 28 male nude mice  were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. . The mice weighed 18–22 g and were raised in a specific pathogen-free environment.7/ml. Under sterile conditions, 0.2 ml cell suspension was inoculated subcutaneously into the nude mice, which were continuously fed for two weeks to establish the nude mouse model. The inoculated mice were randomly divided into four groups, with seven mice in each group; the body weight difference between groups was not significant. In the blank control group, an intraperitoneal injection of saline (10 ml/kg) was performed every other day. In experimental group 1 (the 5-Fu group), an intraperitoneal injection of 5-Fu (60 mg/kg) was administered every other day. In experimental group 2 (the celecoxib group), an intraperitoneal injection of celecoxib (30 mg/kg) was given every other day. In experimental group 3 (the combination group), celecoxib (30 mg/kg) and 5-Fu (60 mg/kg) were administered by injection every other day. All treatments were continued for 2 weeks. The diet, activity, urine and tumor growth of the nude mice were observed every day. At the end of the experiment, the mice were weighed and the tumor size was measured. Mice were sacrificed by cervical dislocation, the tumor was stripped with scissors and the tumor weight was documented. The procedures carried out in the present study were approved by the Medical Ethics Committee of the Affiliated Hospital of Weifang Medical University .A total of 28 male mice (BALB/c nu/nu) aged 5–6 weeks and weighing 18–22 g were used in the experiment. SGC7901 human gastric cancer cells in the logarithmic growth phase were used to create a cell suspension with a concentration of 1×102)/2, where L is the long diameter and S is the short diameter of the tumor.On the day after the final administration, the animals were sacrificed by cervical dislocation, the tumor tissue was excised and the tumor mass was weighed using electronic scales. The inhibition rate in each group was calculated according to the following formula: Inhibition rate (%) = /average tumor weight in control group × 100. The short and long diameters of the tumor were measured and the tumor volume was calculated using the following formula: Tumor volume (V) = (L × SMice xenograft specimens from each group were cut (5 μm) and fixed with 10% formalin. The sections were paraffin-embedded, stained with hematoxylin and eosin and observed under a light microscope. The immunohistochemical staining of tumor tissue from each group was conducted using an SP-9000 kit according to the instructions of the manufacturer. HIF-2α, ABCG2 and Oct-4 antibody staining was carried out (dilution 1:50) following which the tissues were placed in a 4°C refrigerator overnight. 3,3′-Diaminobenzidine color rendering, dehydration, transparency were performed and the tissues were mounted with neutral gum. Phosphate-buffered saline replaced the primary antibody to act as the negative control. Staining for HIF-2α, Oct-4 and ABCG2 was considered to be positive when brown particles were observed within the cytoplasm.The TRIzol method was used for extraction of total RNA from the tumor tissue. The RNA was dissolved in 30 μl diethylpyrocarbonate-treated water. First-strand cDNA was reversely synthesized, using an RT reaction system (20 μl) as follows: 9 μl deionized water with no RNA enzyme, 2 μl template RNA, 181 μl Oligo (dT), 4 μl 5× reaction buffer, 1 μl RNase inhibitor (20 U/μl), 2 μl dNTP mix (10 mmol/l) and 1 μl M-MuLV RT. The reaction conditions were as follows: 70°C for 5 min and then placed on ice; 37°C for 5 min; 37°C for 60 min; 70°C for 10 min and then placed on ice for subsequent testing or preservation at −150°C.2 (25 mmol/l), 5 μl 10× PCR buffer and 34 μl ddH2O were maintained at 94°C for 5 min; 94°C for 30 sec; 50°C for 30 sec and 72°C for 60 sec for 40 cycles. Extension was carried out at 72°C for 10 min and 4°C for +∞. Then, 1.5% agarose gel electrophoresis was used for identification of the product. A digital gel imaging system was used to capture images and the optical density of the amplification products was analyzed. Through analysis of the HIF-2α, ABCG2 and Oct-4 mRNA and GAPDH optical density values, the expression levels of HIF-2α, ABCG2 and Oct-4 mRNA were evaluated.The primers used were HIF-2α, forward: 5′-CTTGGA GGGTTTCATTGCTGTGGT-3′ and reverse: 5′-GTGAAG TCAAAGATGCTGTGTCCT-3′, with a product length of 123 bp; ABCG2, forward: 5′-CCCTTATGATGG TGGCTTATTC-3′ and reverse: 5′-GTGAGATTGACC AACAGA CCAT-3′, with a product length of 132 bp; Oct-4, forward: 5′-CCCGAAAGAGAAAGCGAACC-3′ and reverse: 5′-CAGAACCACACTCGGACCAC-3′, with a product length of 151 bp; and GAPDH, forward: 5′-GCACCACCA ACTGCTTAGCAC-3′ and reverse: 5′-GCAGCGCCA GTAGAGGCAGG-3′, with a product length of 143 bp. In the 50-μl PCR reaction system, 1 μl template cDNA, 1 μl each of upstream and downstream primers, 1 μl Taq DNA polymerase, 5 μl dNTPs (2 mmol/l), 2 μl MgClThe total protein of the tumor tissue was extracted. The bicinchoninic acid assay method was used to determine the protein concentration. SDS-PAGE gel electrophoresis was then conducted. The proteins were placed on a cellulose membrane and sealed with 5% skimmed milk powder at 37°C for 2 h. HIF-2α, ABCG2 and Oct-4 and β-actin antibodies were added , and the membrane was incubated overnight at 4°C. After washing the membrane with Tris-buffered saline and Tween 20, the secondary antibody horseradish peroxidase-labeled anti-IgG (Invitrogen Life Technologies) was added and the membrane was incubated for a further 2 h at 37°C. After washing the membrane, the electroluminescence (ECL) reagent was added. X-ray film exposure, developing and fixing were performed. LabWorks analysis software, version 4.5  was used to measure the absorbance value of the western blotted strip; the ratio of the absorbance value of the protein of interest to that of β-actin was considered to indicate the relative content of HIF-2, ABCG2 and Oct-4.Data were analyzed using the SPSS statistical package, version 17.0 . Quantitative data are expressed as the mean ± standard deviation. Quantitative data were compared between groups using a Student’s t-test and analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant difference.The formation of nodules was observed in the 28 nude mice 14 days after gastric cancer cell inoculation; all grew into a tumor. The average volumes of the tumor mass in the four groups were not significantly different prior to the administration of the various treatments (P>0.05). However, 15 days after the treatment was initiated , the average volume of the tumor mass in the 5-Fu group was less than that in the control group, but the difference was not significant (P>0.05). The average volumes of the tumor mass in the celecoxib group and the combined group were significantly lower than that in the control group (P<0.05), and the average volume of the tumor mass in the combined treatment group was significantly less than the volume in the 5-Fu group (P<005). Similar results were obtained for the tumor weight. The mean tumor weight in the 5-Fu group was not statistically significant different from that in the control group (P>0.05). The mean tumor weights in the celecoxib and combined treatment groups were significantly lower than that in the control group (P<0.05), and the mean tumor weight in the combined treatment group was significantly less than the mean weight in the 5-Fu group (P<0.05). The tumor inhibition rates in the 5-Fu, celecoxib and combination groups were 26.36, 59.70 and 88.37%, respectively. A statistically significant difference was identified between the combination group and the 5-Fu and celecoxib groups .The positive expression of HIF-2α, ABCG2 and Oct-4 proteins was identified as brown granular staining. The proteins were mainly dispersed throughout the cytoplasm and along the nuclear membrane border in a linearly distributed manner, and a strong positive reaction was observed in the nucleus.For the four groups of nude mice after 14 days, the expression levels of HIF, ABCG2 and Oct-4 proteins were highest in the tumor tissue of the 5-Fu group, followed by the blank control group, and the expression levels of the three proteins in the combined and celecoxib groups were significantly reduced –4.The RT-PCR technique was used in the four groups of nude mice to detect the mRNA expression of HIF-2α, ABCG2 and Oct-4 in the tumor tissue after 14 days of treatment. In the control group, which received saline every other day, HIF-2α, ABCG2 and Oct-4 mRNA expression was observed at high levels. In the 5-Fu group, the levels of HIF-2α, ABCG2 and Oct-4 mRNA expression were increased by the injection of 5-Fu every other day. In the celecoxib and combined treatment groups, the HIF-2α, ABCG2 and Oct-4 mRNA expression levels were lower than those in the 5-Fu group. The celecoxib and combination treatment groups showed a significant difference from the control group when a pairwise comparison was performed P<0.01;  and 6.The western blotting technique was used to detect the protein expression of HIF-2α, ABCG2 and Oct-4 in the tumor tissue after 14 days of treatment. The results showed that the HIF-2, ABCG2 and Oct-4 protein expression levels in the tumor tissue were high in the control group, and were further increased in the 5-Fu group. The HIF-2α, ABCG2 and Oct-4 protein expression in the tumor tissue was significantly decreased in the celecoxib and combination treatment groups compared with the control and 5-Fu groups. For all four groups, a pairwise comparison was performed  and 8. TGastric cancer is an disease that is seriously harmful to human health, as it has a high morbidity and mortality. Surgery remains the only mean possible to cure gastric cancer, but in approximately two-thirds of cases, the patient’s condition has reached advanced gastric cancer at the time of diagnosis ,8. It haet al , indicating that a 5-Fu insensitive subpopulation of cancer stem cells is the source of resistance to chemotherapy. The preliminary results of the present study demonstrated that under hypoxic conditions, when 5-Fu was used to treat human gastric cancer cell lines, the expression of cancer stem cell markers HIF-2α and ABCG2 increased (Previous studies have suggested that a very small amount of tumor tissue has unlimited self-renewal capacity and multi-cell proliferative potential, due to the presence of cancer stem cells. These are considered to be the root cause of metastasis, recurrence, drug resistance and chemotherapy resistance ,14. Howeet al  reportedl (et al  demonstrncreased . In the et al (in vivo, specifically, in gastric cancer transplants in nude mice. The inhibition rate in the combination treatment group was significantly enhanced compared with that in the 5-Fu group. Celecoxib and 5-Fu exhibited a synergistic effect in the treatment of gastric cancer. However, the specific antitumor mechanism of NSAIDs remains unclear. Previous cell and animal experiments have shown that NSAIDs inhibit COX-2 activity to reduce the synthesis of prostaglandin E2, thereby inducing tumor cell apoptosis (et al found in a premalignant and malignant oral mucosal cell culture model, that the potential anticancer and apoptosis-inducing effects of celecoxib occurred via a mechanism that was independent of COX pathways (Celecoxib is a selective COX-2 inhibitor that has anti-inflammatory and analgesic effects. Clinically, it is used for the treatment of acute and chronic osteoarthritis and rheumatoid arthritis. Compared with conventional NSAIDs, it has significantly reduced gastrointestinal side-effects. Clinical and experimental studies have shown that celecoxib also plays a role in tumor suppression. Steinbach et al  performeet al ,25. In tpoptosis . Howeverpathways . Numeroupathways –33. The In conclusion, HIF-2α, ABCG2 and Oct-4 mRNA and protein expression levels were significantly increased in the tumor tissues of the 5-Fu group; this may be a due to the tumor cells having resistance to 5-Fu. However, when 5-Fu and celecoxib were used together, compared with 5-Fu used alone, the HIF-2α, ABCG2 and Oct-4 mRNA and protein expression levels were significantly lower, and the difference was statistically significant. This showed that the combined use of 5-Fu and celecoxib is able to attenuate the resistance to chemotherapy in gastric cancer and enhance the effect of chemotherapy by reducing the expression of HIF-2α, ABCG2 and Oct-4 and cancer stem cells in tumor tissue xenografts in nude mice.


Mycobacterium tuberculosis (Mtb) strains using genotyped Mtb isolates from tuberculosis patients is a routine public health practice in the United States. The present study proposes a standardized cluster investigation method to identify epidemiologic-linked patients in Mtb genotype clusters. The study also attempts to determine the proportion of epidemiologic-linked patients the proposed method would identify beyond the outcome of the conventional contact investigation.Tracking the dissemination of specific Mtb culture positive patients from Georgia, Maryland, Massachusetts and Houston, Texas. Mtb isolates were genotyped by CDC’s National TB Genotyping Service (NTGS) from January 2006 to October 2010. Mtb cluster investigations (CLIs) were conducted for patients whose isolates matched exactly by spoligotyping and 12-locus MIRU-VNTR. CLIs were carried out in four sequential steps: (1) Public Health Worker (PHW) Interview, (2) Contact Investigation (CI) Evaluation, (3) Public Health Records Review, and (4) CLI TB Patient Interviews. Comparison between patients whose links were identified through the study’s CLI interviews (Step 4) and patients whose links were identified earlier in CLI (Steps 1–3) was conducted using logistic regression.The study population included Forty-four clusters were randomly selected from the four study sites . Epidemiologic links were identified for 189/401 (47 %) study patients in a total of 201 linked patient-pairs. The numbers of linked patients identified in each CLI steps were: Step 1 - 105/401 (26.2 %), Step 2 - 15/388 (3.9 %), Step 3 - 41/281 (14.6 %), and Step 4 - 28/119 (30 %). Among the 189 linked patients, 28 (14.8 %) were not identified in previous CI. No epidemiologic links were identified in 13/44 (30 %) clusters.Mtb genotype clusters, which can be integrated into the TB control and prevention programs in public health settings. The CLI interview identified additional epidemiologic links that were not identified in previous CI. One-third of the clusters showed no epidemiologic links despite being extensively investigated, suggesting that some improvement in the interviewing methods is still needed.We validated a standardized and practical method to systematically identify epidemiologic links among patients in The online version of this article (doi:10.1186/s12879-016-1937-9) contains supplementary material, which is available to authorized users. Mycobacterium tuberculosis (Mtb) strains in populations is an important tool used to understand TB transmission dynamics . No difference in the number of clusters having 100 % foreign-born patients was seen between the two groups (data not shown).There was substantial variability by cluster in terms of the proportion of patients with identified epidemiologic links . All epidemiologic links with a household transmission setting and/or involving relatives were identified earlier than Step 4, while workplace and church transmission settings were associated with identification through CLI TB patient interviews in Step 4 . Epidemiologic links involving a black TB patient had higher odds of being identified by early investigation steps (p = 0.036). Epidemiologic links including Asians or patients with extrapulmonary TB were associated with identification through CLI TB patient interviews in univariate analysis ; these associations became non-significant in multivariate results. Definite (strength) epidemiologic links had decreased odds for identification through interviews (p < 0.007)  found in the CLI interview were not identified through the previous CI . When applied in a local health department context, existing knowledge of clusters or patient relationships is available through communication with a case manager, disease intervention specialist, or contact investigator . Existing contact investigation records were then reviewed for documented links (Step 2). The next investigation step, entailing review and evaluation of public health records, added a more time-intensive and analytic component to investigations Step 3). Finally, the most resource intensive step was patient re-interviews (Step 4). The analysis of the CLI step where epidemiologic links were determined , the inability to locate and obtain consent from patients for re-interviews and exclusion of clinically defined and non-genotyped culture-positive patients with epidemiologic links to patients in study clusters. In addition, the infectious period of each patient was not considered. Although beyond the scope of this study, including non-genotyped patients may show a more complete picture of cluster transmission dynamics. Furthermore, NTGS transitioned from using spoligotype and 12-locus MIRU-VNTR (MIRU12) to spoligotype and 24-locus MIRU-VNTR (MIRU24) in 2009 to increase the discriminatory power of MIRU-VNTR , 14, 15.Mtb genotype clusters. In addition, CI record review (Step 2) found 15 (3.9 %) linked patients exemplifying a need for better tools and trainings for contact investigations, which is an essential component of TB control programs.Public health departments need to develop strategies and focus resources to prioritize and investigate clusters that may be of public health concern. An initial step in these investigations should be to evaluate clusters using readily available data. Many data elements needed to investigate clusters in specific jurisdictions are now available to TB control personnel routinely and electronically through the Tuberculosis Genotyping Information Management System . AdditioMtb transmission by implementing the expanded and efficient CIs and CLIs would be critical for the success of TB control and prevention programs in the US.Despite the continuing decline in US TB rates leading to a decrease of funding for public health activities for TB control, the elimination goals established in 1989  remain uMtb genotype clusters.We validated a practical method to systematically identify tuberculosis epidemiologic links that can be integrated into routine TB control and prevention programs in public health settings. Re-interviewing patients in a cluster can identify additional epidemiologic links that were not found in the previous CLI steps. Improvement of the interview methods and effective contact investigation trainings may be needed as no epidemiologic links were identified in one-third of the


We show that this approach, despite being more laborious, is a very efficient pipeline for the creation of large libraries of mutants.Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efficiency of the mutagenesis step is the key for fast and economical generation of such libraries. PCR artefacts such as misannealing and tandem primer repeats are often observed in mutagenesis cloning and reduce the efficiency of mutagenesis. Here we present a high-throughput mutagenesis pipeline based on established methods that significantly reduces PCR artefacts. We combined a two-fragment PCR approach, in which mutagenesis primers are used in two separate PCR reactions, with an  The technique permits mapping of protein–protein interaction interfaces, ligand binding sites and residues important for enzymatic activity or general functionality. Furthermore, it allows protein engineering by combination of mutations with desired characteristics. While mutagenesis was initially limited to very few residues, improvement of mutagenesis techniques now enables scanning of an entire protein sequence of several hundred amino acids. This was demonstrated for both soluble and membrane proteins, including several G protein-coupled receptors (GPCRs) with the aim of stabilising the inherently unstable proteins, reviewed in ref. i1 and arrestin-1 residues involved in GPCR signalling5. As an alternative to using scanning mutagenesis based on predefined sets of mutations, random mutagenesis is also a very powerful approach applied to protein engineering and establishing structure-activity relationships10.Scanning mutagenesis, the replacement of selected single amino acid residues by alanine or any other amino acid11) of the widely used QuikChange™ method , both partially overlapping PCR primers contain the mutation which is then inserted in the newly synthesized DNA. The PCR-linearized plasmid is then transformed and repaired (circularized) by homologous recombination in the bacterial cells. Although being very simple, this approach sometimes results in artefacts in the final PCR products13. Several versions of this method are available for producing site-directed mutants, with varying degrees of complexity and efficiency. All of them could be used for library generation. However, when making a large number  of site-directed mutants, and aiming for 100% coverage of the gene of interest, the differences in efficiency become very important for the overall speed, success rate and cost of library generation.Generally, alanine-scanning mutagenesis is based on PCR amplification of the recombinant DNA followed by an enzymatic digest of the methylated template DNA using endonuclease DpnI. In the improved version  and bovine arrestin-1  proteins13. After the initial success of the two-fragment approach in generating several “difficult-to-make” arrestin-1 mutants, we decided to use this strategy for high-throughput scanning mutagenesis of two human GPCRs – cannabinoid CB2 receptor  and vasopressin V2 receptor .The mutagenesis primers are combined with another pair of primers, annealing approximately on the opposite side of the plasmid Fig. . The twoHere, each vector fragment was amplified by PCR using one mutagenic primer and the appropriate ColE1 primer annealing to the complementary DNA strand within the vector’s bacterial origin of replication  or valines (CB2). The alanine and glycine substitutions remove the side chain of the amino acid and are used for probing its role in protein function. Valines are one of the prevalent types of amino acids in membrane proteins and promote stability of transmembrane helices. These substitutions are generally well tolerated and used for thermostabilisation of GPCRs  agar plates to save space without losing efficiency of mutagenesis. Alternatively, bacteria could be plated on 24-well plates using an expanding pipette21. It should be noted that 68 CB2 transformants gave no visible colonies on the selection plates. We hypothesized that the corresponding PCRs were not successful, so we repeated them using another DNA polymerase and the corresponding PCR conditions .After PCR, the two matching fragments carrying one mutation were combined without checking PCR products on an agarose gel Fig. . The metin vivo recombination in the E. coli Mach1 strain22, according to our earlier one-fragment approach13. When few mutants remain to be cloned, we tend to try different approaches in parallel and/or send more clones at once. Therefore, the standard single-fragment approach was used alongside the two-fragment method.Colonies were sent for sequencing on 96-well LB agar plates with the appropriate selection antibiotic. Initially, we sent out only one colony per mutant to reduce sequencing costs. If the first sequencing reaction was not successful, we sequenced DNA from one to three additional colonies. Difficult CB2 mutants 14, 4%) were obtained by repeating PCR, analysing products on agarose gels and cutting the right bands out in case of significant side-products. Alternatively, the rest of the missing V2R mutants  were obtained by repeating the PCR with two mutagenic primers in the same reaction mixture . We use this term when discussing occurrence of primer tandem repeats, which were the main cause of failure in the one-fragment approach used in arrestin-1 high-throughput mutagenesis we use as a comparison. However, it should be noted that sequencing failures could also be caused by unsuccessful cloning, e.g. when a part of plasmid to which sequencing primer should anneal is missing. Because we cannot distinguish a true sequencing failure from a cloning failure in our data, we decided to use an overall success rate of the method as well. It takes into account all encountered practical difficulties and it is defined as a percentage of the expected sequences obtained in all sequencing attempts (including failed sequencing and non-informative sequencing data). In our opinion, this information is valuable in the context of high-throughput site-directed mutagenesis with direct repercussions on expected costs.Taken together, we successfully generated complete alanine scanning libraries of the two GPCRs. For V2R, 467 out of 565 sequencing reactions confirmed the designed single amino acid mutations, which correspond to an overall success rate of 83%. For CB2, we obtained the right sequences for 390 out of 537 colonies sent for sequencing (73%). The lower efficiency in this case was mostly due to failed sequencing or lower quality of sequencing data not allowing their reliable interpretation as well as primer misannealing resulting in large deletions  – two plant membrane proteins with an extensive cytoplasmic domain. Out of 50 successfully sequenced DNA samples, 45 had the correct sequence. Although the remaining 5 had the correct truncation, they also contained an additional point mutation, presumably introduced as a DNA polymerase error in PCR. While the Gibson method is an established cloning method, these results suggest that it is very well suited for general high-throughput applications.We also used the same two-fragment cloning approach to prepare twenty N- and/or C-terminally truncated constructs of 13. In our case, the pCDNA4/TO vector encoding for fusions of the CB2 and V2R constructs used in this work have the same length of 7 kbp and an average GC content of 55 and 54%, respectively . The constructs encoding for the ETR1 were in the range of 6.2–7.5 kbp. Based on the two parameters that can dramatically affect the PCR efficiency, the length and GC content, these samples are relatively similar. The plasmid size is very critical for co-transformation cloning, where overlapping fragments are simply used to co-transform E. coli, without in-vitro treatment with In-Fusion or assembly mix15. However, the assembly mix is efficient at combining DNA fragments up to several hundred kilobases, a size that is never reached with plasmids14. If insert-plasmid mixtures are combined in vitro using assembly mix, as in our method, the limiting factor is the PCR. Because we use two fragments and 6 kb can be robustly amplified today, the method works for plasmids up to at least 12 kb. The larger plasmid size also has a slight negative effect on transformation efficiency, but this is not a problem with competent cells of reasonably high transformation efficiency. In comparison to the method previously used in our lab with both mutagenic primers in one PCR and recombination in Mach1 cells13, we found that a number of instances with tandem repeats of the primer sequence before or after the mutation site was drastically reduced , longer elongation times (at least 30 s/kbp) and less PCR cycles in combination with annealing temperatures not lower than 60 °C . Furthermore, the Hot Fusion methodThe two-fragment approach presented here can be further extended to a three-fragment approach where the whole plasmid backbone is prepared by restriction digestion, mutagenesis is then done with mutagenesis primers and primers flanking the gene of interest, followed by PCR clean-up and Gibson assembly. This could be a very efficient way of making mutants, especially in the GC rich regions that are challenging for the PCR. An additional benefit is that the backbone, since it is only produced once, can be verified for the absence of mutations and deletions, something that is not practical with the two-fragment approach. It needs to be noted that, from our experience, it is more difficult to assemble three fragments successfully, especially when the molar ratio of the assembling fragments has not been chosen carefully. In our two-fragment high-throughput approach, we do not measure or adjust fragment concentration before the assembly reactions.Although there are many general cloning methods developed and kits commercially available, it is of crucial importance to have a highly-efficient, simple, fast and cost-effective method when it comes to generating libraries comprised of hundreds of mutants with 100% coverage. Even though the presented high-throughput mutagenesis and cloning pipeline is slightly more laborious than other established mutagenesis protocols, we found this to be a convenient and efficient alternative.13. The AAscan, PCRdesign and MutantChecker are an open source software written in an open source multi-platform Pascal-Lazarus environment25 and are freely available from the authors’ web site (https://www.psi.ch/lbr/aascan).In this work, we used software specially developed for primer design and sequence analysis in high-throughput scanning mutagenesisTm) of 60–70 °C with a maximum ΔTm of 5 °C for a pair of primers. Our designed primers, excluding the mismatching bases, had annealing temperatures in a range 60–68 °C. The minimal overlap of the two mutagenic primers was 21 nucleotides for CB2 and 15 for V2R. A minimum of 13–15 bases is needed for both Gibson assembly and bacterial recombination to work27. A too long overlap may restrict primer design options of the program, which may lead to a decrease in mutagenesis success rates. In several cases of CB2 mutagenesis, the value of 21 nucleotides had to be decreased in order to design primers within the given restraints. Longer overlap of the primers used for CB2 mutagenesis may be an explanation for the lower success rate compared to V2R mutagenesis. Codons were chosen according to their frequency in the expression host and according to the GC content of the gene. Very high and very low GC contents should be avoided. For mammalian expression vectors, we chose the codons GCC/GCT for alanine, GGC/GGA for glycine and GTG/GTC for valine. For genes with high GC contents it could be advisable to choose GCT/GCA for alanine, GGA/GGT for glycine and GTT/GTA for valine, provided that the host organism is able to translate these codons efficiently. Due to consecutive cytosine bases in the CB2 gene sequence, several primers designed in AAscan had to be manually modified by introducing other degenerate codons in order to avoid G-repeats in the reverse (antisense) primers. G-repeats are prone to formation of G-quadruplex secondary structure  and might thus interfere not only with the oligonucleotide production and quantification, but might also reduce PCR efficiency. Primers used for the C- and/or N-terminal truncations of ethylene receptor 1 from Arabidopsis thaliana  and tomato  in pET16b vectors were designed in program PCRdesign13 with the following parameters: a primer length 16–50 nt, an overlap 21 bp (base pairs), a minimum GC clamp of 2, optimized GC content and a minimum Tm of 60 °C.For human CB2 and V2R genes in pcDNA4/TO vector (both plasmid sizes 7.0 kb) we designed the mutagenic primers in a high-throughput way by using the program AAscan and the following parameters: a primer length 18–60 nt (nucleotides), a minimum GC clamp of 2, a melting temperature , pH 7.5. Other primers were ordered from Microsynth or Sigma-Aldrich in lyophilized form.28. It is also possible that the PCR reaction was not efficient enough to be detected by ethidium bromide staining.PCR conditions were first tested for a random set of 30 CB2 mutants using Phusion High-Fidelity PCR Master Mix with HF Buffer, Phusion High-Fidelity PCR Master Mix with GC Buffer  and KOD Hot Start Master Mix (Merck Millipore). Based on analysis of the PCR products on agarose gels , and either 1× Phusion buffer HF (NEB) or 3% (v/v) DMSO (NEB) with 1× Phusion buffer GC (NEB). Also, PCR thermocycling described above was modified in case of the ETR1 plasmids to have: (1) 11 step-down cycles with annealing starting from 65 °C down to 60 °C (i.e. decreasing by 0.5 °C in each subsequent cycle); (2) elongation time of at least 30 s per kbp of the target product (up to 200 s for the longest PCR products); and (3) final elongation time of 5 min.For the two-fragment approach, each mutagenesis primer was combined with the matching ColE1 primer, giving two PCR products per mutation. For the one-fragment approach, the two mutagenesis primers were combined in one PCR reaction, which required a longer elongation time compared to the two-fragment approach. PCRs were carried out in 20 μL final volume with 1 ng template DNA, 300 nM of each primer and, in case of CB2 only, 3% (v/v) DMSO (dimethyl sulfoxide). Good results were obtained with the following thermocycling setup using Phusion High-Fidelity DNA Polymerase with GC buffer: initial denaturation at 98 °C for 1 min, followed by 20 step-down thermal cycles, each comprising denaturation at 98 °C for 20 s, annealing from 65 °C down to 55.5 °C for 30 s (0.5 °C decrement per cycle), and elongation at 72 °C for at least 25 s per kbp of the expected PCR product , then 10 thermal cycles with the constant annealing temperature , and final elongation at 72 °C for 3 min completed with a hold at 10 °C. When using KOD Hot Start Master Mix, the following thermocycling setup was used: Initial denaturation at 95 °C for 2 min, followed by 20 step-down thermal cycles, each comprising denaturation at 95 °C for 20 s, annealing from 65 °C down to 55.5 °C for 10 s (0.5 °C decrement per cycle), and elongation at 70 °C for at the appropriate time (up to 135 s), then 5 thermal cycles with the constant annealing temperature , completed with a hold at 10 °C. For truncations of the AtETR1 and LeETR1 plasmids by the two-fragment approach, a slightly different PCR protocol was used: 20 μL PCR mixture prepared with nuclease-free water  contained 1 ng DNA template, 500 nM each primer, 200 μM each dNTP , 0.4 U μL−1 from NEB or 10 units μL−1 from Thermo Scientific) to a final PCR mixture. The mixtures with DpnI were incubated at 37 °C for 18 h (GPCR mutagenesis) or 2 h (ETR1 truncated products), however the digestion time can be shortened if necessary, provided the template amount in PCR and DpnI amount are optimised beforehand. The reaction was cleaned-up using a 96-well ZR-96 DNA Clean-up Kit (Zymo Research), MinElute Reaction Cleanup Kit (Qiagen) or Illustra GFX PCR DNA and Gel Band Purification Kit  according to the manufacturer’s instructions. We used a low elution volume of 10 μL to obtain higher DNA concentrations and, in case of two-fragment cloning, a more efficient Gibson assembly.After PCR, the two fragments carrying one mutation in a GPCR gene were combined already before digestion and purification. To reduce background, methylated template DNA was digested by adding 0.5 μL DpnI enzyme (20 units μLin vitro using Gibson assembly14. Twelve mL 1.33× Gibson assembly mix was prepared in-house using 6.4 μL 10 U μL−1 T5 exonuclease (NEB), 200 μL 2 U μL−1 Phusion High-Fidelity DNA polymerase (NEB) and 1.6 mL 40 U μL−1 Taq DNA ligase (NEB) in isothermal reaction (IT) buffer (5% (w/v) PEG-8000 (poly(ethylene glycol) 8000), 100 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM DTT (dithiothreitol), 1 mM β-NAD (β-nicotinamide adenine dinucleotide), 200 μM of each dNTP). IT buffer was prepared as a 5× stock in nuclease-free water. In case of the CB2 and V2R mutants, 1 μL cleaned-up DNA mixture was added to 3 μL 1.33× Gibson assembly mix and incubated at 50 °C for 10 min followed by 1 h at 37 °C. In case of CB2, with 21 bp overlap within a pair of mutagenic primers, we observed more colonies on selective LB agar plates when Gibson reaction was performed for 1 h at 50 °C. For ETR1 truncations, 2.5 μL 1.33× Gibson assembly mix was combined with 1 μL of the longer PCR product and 1.5 μL of the shorter one, without determining DNA concentration of each cleaned-up PCR product, and the assembly was conducted at 50 °C for 1 h.Cleaned PCR products with two fragments were assembled Escherichia coli XL-1 Blue cells were prepared by the Inoue method30 and had a transformation efficiency of at least 107 colonies per 1 µg plasmid pBR322. The assembled fragments were transformed into the competent cells using 20 μL of cell suspension and 2 μL assembly mix for GPCR mutagenesis or 50 μL cell suspension and 5 μL assembly mix for ETR1 mutagenesis. Cells for transformations were thawed on ice, aliquoted, incubated with DNA for 10–20 min, heat shocked for 1 min at 42 °C, incubated on ice for 2 min and plated on LB agar plates with 150 μg mL−1 ampicillin. A quarter of LB agar plate was used for each V2R mutant, while the transformed cells of other constructs were spread over a whole plate. Plated cells were incubated at 37 °C for 16–24 h.Chemically competent PCR reactions, heat shock for transformation and Gibson assemblies were done in 96-well PCR plates (Eppendorf) using Mastercycler pro S or Mastercycler gradient thermocyclers (Eppendorf).−1 ampicillin and sent for plasmid preparation and sequencing by the GATC Biotech Company. Sequencing results were analyzed with the program MutantChecker13. In case the mutant was not obtained in the first round, another clone was sent for sequencing. If there were no more clones, PCR with modified conditions and/or Gibson assembly was repeated. ETR1 plasmids were prepared using NucleoSpin Plasmid Kit (Macherey-Nagel) and sequenced by the Biological-Medical Research Centre (BMFZ) at the Heinrich Heine University Düsseldorf.In the high-throughput CB2 and V2R mutagenesis, one cell colony per mutation was transferred into a well of a 96-well LB agar plate with 150 μg mLA small number of mutants have proven difficult to make, 14 (4%) in case of CB2 and 3 (0.8%) in case of V2R. In the CB2 mutagenesis, a large deletion in the plasmid was a most common problem. This is a clear indication of a primer misannealing in PCR, probably related to a higher GC content (61%) in the CB2 gene, while no correlation with high GC content in the primer itself was observed (Supplementary Data). To obtain missing mutants, PCR was repeated using different DNA polymerase (Hot Start Master Mix). In the third round of mutagenesis, PCR was repeated using both Phusion High-Fidelity DNA Polymerase with GC buffer and KOD Hot Start Master Mix with the conditions as described above, but longer elongation times were used (135 s). PCR fragments were analysed on agarose gels . Linear PCR products were digested with DpnI and directly transformed into the chemically competent E. coli Mach1 strain for in vivo recombination, in which case SOC medium was added after transformation followed by 2 h recovery at 37 °C to allow bacterial DNA repair before plating on LB agar plates with the selective antibiotic.An alternative approach was used for obtaining the three remaining V2R mutants. In this case, a PCR was repeated, but with the two mutagenic primers in the PCR mixture (“single-fragment” approach) and a longer extension time which is sufficient for full plasmid amplification (Sun, Ostermaier The results of data analysis are included in the supplementary information.Supplementary Dataset 1


A 40-year-old female patient with a 5-year history of systemic lupuserythematosus was referred to our policlinic with complaints of erythema,atrophy, and telangiectasia on the upper eyelids for 8 months. No associatedmucocutaneous lesion was present. Biopsy taken by our ophthalmology departmentrevealed discoid lupus erythematosus. Topical tacrolimus was augmented to thesystemic therapeutic regimen of the patient, which consisted of continuousantimalarial treatment and intermittent corticosteroid drugs. We observed noremission in spite of the 6-month supervised therapy. Periorbital discoid lupuserythematosus is very unusual and should be considered in the differentialdiagnosis of erythematous lesions of the periorbital area.. Delayed diagnosis usually reflects intreatment delays.3Discoid lupus erythematosus (DLE) is the most common chronic form of lupuserythematosus (LE). DLE is more common than the systemic form of the lupuserythematosus. Clinical features include lesions with squamous and erythematousscaly plaques that may result in atrophic scars, alopecia, or permanent pigmentarychanges. The most commonly involved areas are those exposed to sun, such as theface, the "V" region of the neck, and the extensor sides of the arms; periorbitallocalization is rare.We report a case of DLE on the eyelid in a patient who had been followed fortreatment-resistant systemic LE.A 40-year-old woman had been followed with the diagnosis of systemic lupuserythematosus (SLE) for 5 years in our rheumatology clinic. She presentedphotosensitivity, synovitis, arthritis, hemolytic anemia, leucopenia, positiveantinuclear antibodies, and positive anti-dsDNA antibodies. According to TheAmerican College of Rheumatology (ACR) and the SLICC (Systemic Lupus CollaboratingClinics) criteria, the patient was diagnosed with SLE. She presented to ourdermatology clinic with the complaints and signs of erythema, telangiectasia, andmild atrophy of her upper eyelids and left lower eyelid. The complaints about theeyelids had started about 8 months before the patient sought treatment and worsenedwith sun exposure. Ophthalmologists suspected discoid lupus erythematosus.Ophthalmic examination revealed squamous, erythematous, mildly edematous, andatrophic areas. Scaly patches and telangiectasias were seen on both eyelids (5-6 mmx 1-2 mm on the right upper side and 2 mm x 1 mm on the left upper and lowereyelids) . The lesRoutine tests showed normal results. Laboratory investigation yielded positiveresults for anti-nuclear antibodies and anti-dsDNA tests. We took an excisionalbiopsy from the right upper eyelid based on a suspected DLE diagnosis. Histologicalexamination revealed epidermal atrophy, vacuolar degeneration, and lymphocyticinfiltrations in deep and superficial perivascular and periadnexial areas,confirming the DLE diagnosis . Thepat7 The disease typically manifests with bilateraleyelid involvement and mucocutaneous lesions.2 Cases involving the conjunctiva and the cornea have alsobeen reported. DLE patients presenting with unilateral eyelid involvement are veryunusual and its diagnosis is a challenge for physicians since it lacks the typicalmorphologic signs of CCLE.5 DLE patients may complain of mild pruritus or sometimes pain,but most cases are asymptomatic. Serological and hematological signs are seen inpatients with typical involvement.Discoid lupus erythematosus (DLE) is the most common form of chronic cutaneous lupuserythematosus (CCLE) and is mostly seen in women 20-40 years of age. Eyelidinvolvement is seen in 5%-6% of DLE patients. 8 Some patients presenting with thesesymptoms received treatment for chronic blepharitis or eczema for many years, asillustrated by Aubaret et al. and Duke-Elder etal., who reported cases of DLE presented as chronicblepharitis.10 In our case, the diagnosis wasmade 6 months after the beginning of the treatment.Delay in diagnosis can lead to eye or eyelid complications – such as periorbitaledema, epiphora, trichiasis, conjunctivitis, stromal keratitis, madarosis,ectropion, and entropion. Such complications, in addition to the development of apermanent scar, can disturb the psychological status of the patient.10Several diseases may be initially considered in the differential diagnosis of DLE:basal cell carcinoma, squamous cell carcinoma, Bowen's disease, actinic keratosis,contact dermatitis, atopic dermatitis, seborrheic dermatitis, psoriasis, andsarcoidosis.4 In our case, we observed no healing with the use ofsystemic antimalarial, topical corticosteroid, or topical tacrolimusmedications.Treatment of DLE lesions on the eyelid usually involves oral hydroxychloroquine,topical corticosteroid, and avoidance of sun exposure. Some researchers also suggestthe use of topical tacrolimus or intralesionary steroid. Although DLE on the eyelids typically has a benign course, diagnosis is frequentlydifficult, resulting in patients receiving treatments for different diagnoses. As aconsequence, treatment results may be negative.