Caenorhabditis elegans. Here, we identify bioinformatically F28F8.5, an uncharacterized predicted protein, as the nematode homologue of MED28. As in other Metazoa, F28F8.5 has dual nuclear and cytoplasmic localization and plays critical roles in the regulation of development. F28F8.5 is a vital gene and its null mutants have severely malformed gonads and do not reproduce. F28F8.5 interacts on the protein level with the Mediator subunits MDT-6 and MDT-30. Our results indicate that F28F8.5 is an orthologue of MED28 and suggest that the potential to link cytoplasmic and nuclear events is conserved between MED28 vertebrate and nematode orthologues.The evolutionarily conserved Mediator complex is a critical player in regulating transcription. Comprised of approximately two dozen proteins, the Mediator integrates diverse regulatory signals through direct protein-protein interactions that, in turn, modulate the influence of Mediator on RNA Polymerase II activity. One Mediator subunit, MED28, is known to interact with cytoplasmic structural proteins, providing a potential direct link between cytoplasmic dynamics and the control of gene transcription. Although identified in many animals and plants, MED28 is not present in yeast; no bona fide MED28 has been described previously in  Drosophila indicated that some subunits are critical only for specific gene transcription from endogenous genes but not for transcription from synthetic promoters . The Mediator complex is found in all eukaryotic organisms. The core Mediator complex is comprised of 21 protein subunits in yeast and a similar number (26) in mammals, all named MED followed by a unique numerical designation . In addiromoters . This diromoters .One of the Mediator complex subunits, MED28, is only found in higher eukaryotes. MED28 was originally identified as a gene expressed in endothelial cells where it was named EG-1  ; it was Mediator-28) in nematodes  and NCBI (https://www.ncbi.nlm.nih.gov) databases were searched with BLAST, PSI-BLAST ::gfp::let858(stop)::SEC::F28F8.5P—edited F28F8.5 disrupted by gfp and self-excising cassette (SEC)) and one WT allele of F28F8.5. This line segregates mutant animals.KV3: (8418)—heterozygous animals carrying one edited disrupted allele of F28F8.5 carrying gfp::F28F8.5 in its normal genomic position (F28F8.5(V:15573749)::gfp::F28F8.5P on both alleles.KV4: (8419)—edited F28F8.5 (consisting of approximately 400 bp of the predicted promoter region preceding the coding region of F28F8.5). The gene encoding GFP was amplified from the pPD95.75 vector with primers 6232 and 6233. The complete construct was amplified with primers 7887 and 6234. The resulting fusion construct contains the 3′ UTR from pPD95.75 . The PCR mixture was injected into the gonads of young adult hermaphrodite animals together with marker plasmid pRF4. The sequences of all primers used in the paper are in For preparation of transgenic lines encoding F28F8.5::GFP from extrachromosomal arrays under regulation of endogenous promoter, we used the PCR fusion-based technique . PrimersF28F8.5 gene was edited by insertion of a construct including the coding sequence of GFP and a SEC containing the sqt-1(d) gene (a visible selection marker leading to a Rol phenotype), hs::Cre (heat shock inducible Cre recombinase) and hygR (hygromycin resistance) genes. The sgRNA sequence was targeted near the start of the coding sequence for the F28F8.5 gene using a modified pJW1219 plasmid  as the Cas9 vector (pMA007); it was prepared by PCR with primers 8403A and 8333 and used in a concentration of 50 ng/μl for microinjections. The plasmid pMA007 was co-injected with the rescue repair template plasmid based upon modified pDD282 vector (pMA006) in a concentration of 10 ng/μl and with three markers (see below). The repair template plasmid pMA006 was prepared in two steps. First the plasmid pMA005 was prepared from gDNA of F28F8.5 (containing both repair arms) and amplified by PCR with primers 8404 and 8405 and cloned into pCU19 backbone. The plasmid pMA005 was subsequently modified—the FP-SEC segment was added and the CRISPR/Cas9 site was altered to protect against Cas9 attack. The linear PCR product of pMA005 was prepared using primers 8406 and 8407 with overlapping regions for Gibson assembly . The primer 8406 was prepared with alternate codons for protection against CRISPR/Cas9 site. Linear insert of FP-SEC was prepared by PCR from pDD282 plasmid  with primers 8408 and 8409. Primers were prepared with overlapping parts for cloning into linear pMA005 plasmid by Gibson assembly and the final rescue plasmid pMA006 was prepared. Plasmids pGH8 (10 ng/μl), pCFJ104 (5 ng/μl), and pCFJ90 (2.5 ng/μl)  were used as fluorescent co-injection markers. After microinjections the population of nematodes were grown for three days at 25 °C and hygromycin (Invitrogen) was added in a final concentration of 250 μg/ml. After three days integrated nematodes were selected according to the rolling phenotype and loss of extrachromosomal arrays.Lines with edited genomes were prepared from wild type N2 animals using the CRISPR/Cas9 system as described . Using tF28F8.5 gene with an inserted gfp regulated by the endogenous promoter of F28F8.5 in one allele and one WT allele. This line segregated homozygous animals for F28F8.5:: F28F8.5::gfpP (edited F28F8.5 with SEC—F28F8.5 (V:15573749)::gfp::let858(stop)::PSEC::F28F8.5) with disrupted F28F8.5 on both alleles and expressing GFP under the regulation of the endogenous promoter. Animals of this line were clearly distinguishable by their developmental phenotypes, weak expression of GFP in the cytoplasm and the presence of rol marker. These animals were sterile and had severe developmental defects (see Results). The genotypes were confirmed by single worm PCR of representative animals after their microscopic analysis (with primers 8398 and 8414).Using this strategy, we obtained a heterozygous line (KV3) with a disrupted F28F8.5 gene in the form of gfp::F28F8.5 in its normal genomic position (edited F28F8.5 with gfp tagged to the N—terminus—F28F8.5(V:15573749)::gfp::F28F8.5P).The excision of the SEC was achieved by a 4 h heat shock at 34 °C. The line KV4 was obtained: animals with both alleles carrying the edited gfp was confirmed by single nematode PCR with primers 7887 and 8454, 8398 and 8454. The PCR products were purified and sequenced with primers 8455 and 8456. PCR was done by REDTaq ReadyMix PCR reaction  or by Phusion High-Fidelity DNA Polymerase . During the maintenance of the heterozygous line KV3, animals with one edited disrupted F28F8.5 allele and one allele with edited F28F8.5 after self-excision of SEC were also generated . Schemes for genome editing are accessible in The presence of the knock-in of F28F8.5 cDNA was prepared from total cDNA using primers 7889 and 7890. The plasmid pPCRII(Topo)  containing F28F8.5b cDNA was linearized using restriction enzymes NotI/SacI. The dsRNA was prepared by in vitro transcription using SP6/T7 Riboprobe® in vitro Transcription Systems  from opposing promoters synthesizing complementary single stranded RNA (ssRNA) for both strands of F28F8.5 cDNA and its complementary strand. After in vitro transcription (∼2 h) equal volumes of sense and antisense RNA were mixed, incubated at 75 °C for 10 min and slowly cooled to room temperature during 30 min. Control RNAi was prepared from the promoter region of nhr-60 as previously described . The plasmids were injected into the gonads of young adult hermaphrodites as described .F28F8.5 expressing gfp::F28F8.5 was performed using an inverted Leica SP8 TCS SMD FLIM system equipped with a 63 × 1.2 NA water immersion objective, a pulsed white light laser (470–670 nm), AOBS and two internal hybrid single photon counting detectors, and operated by Leica Application Suite X program . The GFP fluorescence was excited at a wavelength of 488 nm and the emitted light was simultaneously recorded in two spectral ranges .Fluorescence microscopy and Nomarski optics microscopy were done using an Olympus BX60 microscope equipped with DP30BW CD camera . Animals were analyzed on microscopic glass slides with a thin layer of 2% agarose and immobilized by 1 mM levamisole . Confocal microscopy of live homozygous animals with edited https://www.picoquant.com/images/uploads/page/files/14528/technote_tttr.pdf. Data structure, program description and user instructions are also freely accessible at https://github.com/PicoQuant/PicoQuant-Time-Tagged-File-Format-Demos/blob/master/PTU/Matlab/Read_PTU.m. The signal from both time synchronized channels was added up. The false color scale (1–3 ns) is based on the average photon arrival time, with blue color representing short lifetime and red color long lifetime fluorescence.For FLIM acquisitions the single photon counting signal from the internal hybrid detectors, acquired during confocal acquisitions, was simultaneously processed by HydraHarp400 TCSPC electronics  and information about the arrival times of all photons was stored to a hard-drive in TTTR data format. TTTR is freely accessible at 2, 0.45% (v/v) NP40 (Nonidet P-40), 0.45% (v/v) Tween-20, 0.01% (w/v)). Proteinase K was diluted immediately before use as a 20 mg/ml stock solution which was kept on ice and diluted to final working solution at a concentration of 60 μg/ml. The tube was sealed in bottom-up position and the sample transferred to the bottom of the tube by centrifugation. The tube was frozen for 10 min at −70 °C. Next, the tube was heated for 1 h at 65 °C and additional 15 min at 95 °C. The resulting sample was used immediately for amplification of DNA by PCR or stored at −80 °C before further analysis. Similarly, genomic DNA was prepared from selected nematode culture plates and used for further screening by PCR and sequencing.Single animal PCR was used for verification of all transgenic lines. Following the microscopy examination, selected animals were removed from microscopic slides and transferred into caps of PCR tubes with 4 μl of solution of Proteinase K (20 mg/ml) diluted 1:333 in Barstead Buffer  were analyzed by single worm PCR with primers 7887 (sense primer) and 7890 or 8454 (antisense primers).Similarly, homozygous animals with edited F28F8.5 from homozygous animals with the edited disrupted gene, five adult homozygous mutant animals recognized by the phenotype and the same number of young WT hermaphrodites with minimum number of formed embryos were manually harvested and collected in separate Eppendorf tubes. Reverse transcription was done using the Maxima H Minus cDNA synthesis kit  as recommended by manufacturer. Universal probe library and primers designed with the help of ProbeFinder Assay Design Software were used and qPCR was run on LightCycler 2.0 purchased from Roche . An average of three sample cDNAs and three control cDNAs were analyzed (twice in duplicates and one time as single experiments), all containing the same amount of RNA for RT for each experiment. The expression of F28F8.5 was normalized to ama-1 and the values obtained in homozygous mutant animals with disrupted F28F8.5 gene were compared to values obtained in control WT N2 animals.For quantitative RT-PCR, the technique described by mdt-6 was amplified using primers 8292 and 8293 from cDNA prepared from mixed stages C. elegans cultures and cloned into pTNT vector  using the Quick Ligation Kit  and expressed in the rabbit reticulocyte TNT-system . The in vitro transcribed protein was labeled using 35S Methionine . F28F8.5 coding sequence (amplified using 8255 and 8256 primers with 15 bp overhangs for insertion into the vector) was cloned into pGEX-2T vector , amplified with primers 8253 and 8254) using the GeneArt Seamless PLUS Cloning and Assembly Kit , transformed into BL21 Escherichia coli cells and the production of protein was induced by isopropyl β-d-1-thiogalactopyranoside (IPTG) . The Mediator subunit MDT-30 was amplified from mixed stages C. elegans cDNA with sense primer 8302 and reverse primer 8527 (containing FLAG sequence), cloned into pET28a(+) vector , amplified with primers 8519 and 8520) using the Quick Ligation Kit (New England Biolabs), transformed into BL21 E. coli cells and induced by IPTG. The lysate from bacteria producing His6-MDT-30-FLAG was used directly or purified on HiTrap Chelating HP column . Proteins produced by the TNT system or bacterial lysates of bacteria transformed with FLAG labeled Mediator subunits were incubated with glutathione–agarose  adsorbed with equal amounts of GST or GST-F28F8.5. Radioactively labeled proteins were detected using TRI-CARB 1600TR, Liquid Scintillation Analyzer .Binding studies were done as described  with mod35S-MDT-6 was visualized by autoradiography and subsequently, the gel containing radioactively labeled protein was localized using superimposed autoradiograms, excised and the radioactivity determined in the scintillation detector. FLAG-labeled MDT-30 was determined by Western blot using an anti-FLAG antibody ) and quantified densitometrically by ImageJ computer program (https://imagej.nih.gov/ij/download.html) (The resulting samples (labeled proteins bound to GST- or GST-F28F8.5) were separated by polyacrylamide gel electrophoresis. ad.html) .C. elegans homologue of MED28, we queried protein databases with curated SwissProt sequences from UniProtKB. They comprised several mammalian and insect proteins . The more sensitive profile-to-profile HHblitz and HHpred algorithms provided hits to a C. elegans annotated protein F28F8.5a and b with highly significant E-values. According to Wormbase (WS248), two protein isoforms are produced from the F28F8.5 gene, isoform a with the length of 200 amino acids and isoform b that has a two amino acid insertion at position 20 of the N-terminal evolutionarily non-conserved region. The best results were obtained when pre-aligned vertebrate and insect MED28 homologues were used as query in three iterations (E < 10−48 and the probability of true positive >99.99%). When the pre-aligned nematode sequences homologues to F28F8.5 were used to query profiles of human or Drosophila sequences in reciprocal searches, MED28 proteins were obtained with equally significant scores. BLAST and PSI-BLAST searches in their standard settings were not able to reveal a significant hit (E < 10−3); the only nematode hit was a Trichinella spiralis protein (E5RZQ1). However, when the searches in protein databases were limited to sequences from Ecdysozoa with Insecta excluded (conservative inclusion threshold E < 10−6) in the first two iterations and then continued in the complete database of sequences from all species in the subsequent iterations, the final hits of F28F8.5 included human and Drosophila MED28. PSI-BLAST with T. spiralis query sequence in database limited to Ecdysozoa in the first two iterations provided both human and Drosophila MED28 and F28F8.5 in one run (E < 10−8). We concluded from these searches that F28F8.5 is a homologue of MED28 and very likely its previously unrecognized orthologue.To identify the Drosophilae or Caenorhabditae sequences. Only the central core of about 110 amino acids is preserved in metazoan evolution. All PSI-BLAST MED28 homologues possess variable N- and C-termini of 3–80 amino acids showing no conservation across Metazoa. This conservation is loose even just within F28F8.5 gene can be expressed as both an individual and multigene transcript, located as the last gene in a four gene operon that is both SL-1 and SL-2 trans-spliced. To determine the intracellular localization of F28F8.5, we edited the F28F8.5 gene using CRISPR/Cas9 technology. We inserted the gene coding for GFP directly in front of the first codon. The arrangement used in our experiment , the endogenous locus had an N-terminus GFP-tagged F28F8.5 gene that we maintained as homozygous animals, demonstrating this edited allele is fully functional. Note that both known protein isoforms of F28F8.5 (a and b) would be tagged on their N-terminus with GFP by this method.After removal of the SEC from this edited gfp::F28F8.5 and mitotic as well as meiotic nuclei accumulated GFP::F28F8.5 protein  resulted in defective development that was most pronounced in late larval stages. The phenotypes included a dumpy phenotype (Dpy)  , irregulpe (Dpy)  and 5M ape (Dpy)  and 5L. pe (Dpy) –5L and oF28F8.5 expression in homozygous mutant animals  from thexcision .F28F8.5 and one WT allele were grossly normal and produced viable embryos. Unlike in homozygous animals carrying the excised SEC allele, the GFP fluorescence was mostly cytoplasmic and most nuclei were not showing accumulation of GFP. In some embryos, however, the nuclei accumulated GFP indicating probable spontaneous SEC self-excision  in comparison to GST alone, as revealed by Western blot using an anti-FLAG antibody , the close sequence similarity of F28F8.5 to MED28 detected informatically in nematode genomes, a conserved dual nuclear and cytoplasmic expression pattern, and its involvement in a wide range of developmental processes. Thus we suggest F28F8.5 be identified as MDT-28, with the original gene for MDT-28 now recognized as the nematode homologue of perilipin, now named PLIN-1 .Saccharomyces cervisiae and in Candida albicans   . Althougq.phtml)  that alsmdt-30, namely a Dpy, burst through vulva and locomotion defect but not a germline defect . The gene mdt-30 is organized in an operon together with F44B9.8 which is an ortholog of human RFC5 (replication factor C subunit 5) and its inhibition by RNAi leads to embryonic defects. Similarly as F28F8.5, mdt-30 is likely to be expressed independently from the operon since it is trans-spliced with both SL1 and SL2 splice leaders .We propose that MED28 is a candidate Mediator complex subunit linking cytoplasmic structural signals towards the core of transcription regulation. The connection between cytoplasmic events and regulation of gene expression can be seen frequently. Numerous transcription factors are regulated by their spatial restriction, binding or incorporation into cytoplasmic structures and organelles. Many proteins that have primarily cytoplasmic structural functions were shown to possess transcription regulating activity  based on high throughput screens  and TRERF1 . RNAi experiments have been reported for atx-3 and F28F8.9, of which only inhibition of atx-3 produced embryonic arrest in 10–25% of embryos. This suggests that even if our gene disruption is affecting other genes in the operon  (http://www.proteinatlas.org)  ; Human Plas.org) ). F28F8.las.org) .C. elegans. We did not observe elevated cytoplasmic expression of GFP::F28F8.5 in the anchor cell described by Our experiments with transgenes fused to GFP also show the differences between the expression of N- or C-terminally labeled F28F8.5. The expression of fusion transgenes is not entirely without functional and developmental consequences. N-terminally labeled F28F8.5 is likely to be able to maintain the nuclear functions of F28F8.5. It is also able to support, at least partially the cytoplasmic functions of F28F8.5 in male rays, since they are formed but are not entirely normal and defects in some animals were observed. It seems likely that GFP-labeled subunits in viable lines may help localize the place of action of labeled proteins as well as their function in The direct link between effector proteins and the regulation of transcription can be traced to Eubacteria and Archaea. Lrp/AsnC proteins, metabolic effectors in Archaea and related Lrs14 proteins are serving as multipotent (Lrp) and specific (Asn) regulators of gene expression. Lrs14 has a clear negative autoregulatory potential illustrating the ancient origin of the transcriptional function of effector proteins . SimilarIn conclusion, MED28 homologues in vertebrates, insects, and nematodes share similarities indicating their conserved roles in cytoplasmic and nuclear events. It can be hypothesized that many proteins that are primarily building blocks of cellular structures and structure-associated proteins are likely to be part of regulatory loops that regulate gene expression. Similarly, as is the case of evolution of operons in Rhabditida that are formed during evolution if they are biologically tolerated for the sake of other regulatory or energetic gains , regulat10.7717/peerj.3390/supp-1Supplemental Information 1Sequences of MED28 homologues shown in Click here for additional data file.10.7717/peerj.3390/supp-2Supplemental Information 2Panels A, C, E and G show images in Nomarski optics and corresponding panels B, D, F and H in GFP fluorescence. In the twofold stage , the transgene is expressed in epidermal cells–seam cells (panels B and D). The transgene is localized both in the cytoplasm and nuclei  or is found accented in nuclei . In the threefold embryo (panels E and F), the transgene is progressively localized in nuclei (arrowheads) as well as in the cytoplasm of epidermal cells. Panels G and H show a threefold embryo before hatching with the expression of the transgene predominantly in the cytoplasm of intestinal cells (arrow). Panels I (Nomarski optics), J (GFP fluorescence) and K (brightfield microscopy together with recorded GFP fluorescence) show a L3 larva in which the nuclear localization of F28F8.5::GFP becomes more accumulated in nuclei of enterocytes (arrows). Panels L, M and N show an adult hermaphrodite animal with F28F8.5::GFP fluorescence in nuclei of enterocytes and in the excretory cell and its channels (arrows). Panels O to Q show the proximal part of the body of a hermaphrodite in L3 stage in confocal microscopy  and an image in Nomarski optics (panel O). Upper arrows indicate the excretory channels and the lower arrow points to the the body of the excretory cell (in panel Q). Arrowheads indicate nuclei of enterocytes with accumulated F28F8.5::GFP surrounding large nucleoli. F28F8.5::GFP is also localized diffusely in the cytoplasm of enterocytes. Bars represent 10 μm.Click here for additional data file.10.7717/peerj.3390/supp-3Supplemental Information 3Panels A and B show part of the body of an adult hermaphrodite in focus on epidermal cells in Nomarski optics (A) and GFP fluorescence (B). Arrowheads mark GFP signal in nuclei of epidermal cells in panel B. Panels C to L show two L3 larvae (one in panels C to F and second in panels G to L). Panels C, E, G, I and K are in Nomarski optics and correspond to panels D, F, H, J and L in GFP fluorescence in the same focal planes. Pharyngeal cells shown in panel D express GFP::F28F8.5 predominantly in nuclei (marked by an arrow). Panels F, H, J and L show cells of the developing vulva expressing GFP::F28F8.5 predominantly in nuclei shown in 3 focal planes (marked by arrows). Bars represent 50 μm.Click here for additional data file.10.7717/peerj.3390/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.3390/supp-5Supplemental Information 5gfp::F28F8.5 edited gene were observed in Nomarski optics (panels A and D), GFP fluorescence (panels B and E) and DAPI staining (panels C and F). The head area with the pharynx (indicated by long arrows with Ph) is showing nuclei of pharyngeal muscle cells labeled by both GFP fluorescence (B) and DAPI fluorescence (C). Short arrows in panels A, B and C indicate two large nuclei of enterocytes with labeled areas by both GFP fluorescence (B) and DAPI fluorescence (C). Similarly, the neurons of the neuronal cord have nuclei positive in both GFP fluorescence (B) and DAPI fluorescence (C) marked by arrowheads. Panels D, E and F show an adult hermaphrodite in focus on epidermal cells. Arrowheads mark nuclei of epidermal cells positive in both GFP fluorescence (E) and DAPI fluorescence (F). Bars represent 50 μm.Homozygous hermaphrodites carrying Click here for additional data file.10.7717/peerj.3390/supp-6Supplemental Information 6Unprocessed images that were used for the preparation of Click here for additional data file.10.7717/peerj.3390/supp-7Supplemental Information 7snapgene.com).Scheme of the repair template plasmid pMA006 designed using SnapGene software .Scheme of the repair template plasmid pMA007 designed using SnapGene software .Scheme of the modified genomic region of Click here for additional data file.10.7717/peerj.3390/supp-10Supplemental Information 10F28F8.5 genome editing designed using SnapGene software .Scheme of Click here for additional data file.10.7717/peerj.3390/supp-11Supplemental Information 11F28F8.5 in homozygous adult hermaphrodites with the edited disrupted gene. Five adult mutant animals recognized by the phenotype and the same number of WT hermaphrodites with minimum number of formed embryos were manually harvested and collected in separate Eppendorf tubes (three collections each). Extracted RNA was reverse transcribed using the Maxima H Minus First Strand cDNA Synthesis Kit . The aliquots were assayed by quantitative PCR using Roche LightCycler 2.0 as recommended by the manufacturer. The table shows the average of 3 experimental samples and 3 control samples, all containing the same amount of RNA for initial RT and normalized to ama-1.Results of the assessment of the level of expression of Click here for additional data file.


C–S–A gene system determines rice hull pigmentation. A conserved color-producing model reveals the independent origin and evolution of the anthocyanin biosynthesis pathway in rice.The  Oryza sativa) can be purple, brown, or red in color due to the accumulation of flavonoids, but the molecular mechanism underlying specific organ pigmentation is not clear. Here, we propose a C–S–A gene model for rice hull pigmentation and characterize it through genetic, molecular, and metabolomic approaches. Furthermore, we conducted phylogenetic studies to reveal the evolution of rice color. In this gene system, C1 encodes a R2R3-MYB transcription factor and acts as a color-producing gene, and S1 encodes a bHLH protein that functions in a tissue-specific manner. C1 interacts with S1 and activates expression of A1, which encodes a dihydroflavonol reductase. As a consequence, the hull is purple where functional A1 participation leads to high accumulation of cyanidin 3-O-glucoside. Loss of function of A1 leads to a brown hull color due to accumulation of flavonoids such as hesperetin 5-O-glucoside, rutin, and delphinidin 3-O-rutinoside. This shows a different evolutionary pathway of rice color in japonica and indica, supporting independent origin of cultivars in each subspecies. Our findings provide a complete perspective on the gene regulation network of rice color formation and supply the theoretical basis for extended application of this beneficial trait.Floral organs in rice ( As major secondary metabolites, flavonoids are well known for giving distinctive floral organ colors and as antioxidants with beneficial effects on human health . A conseC1/P1 and R/B genes, which encode R2R3-MYB and bHLH transcription factors, respectively (P1 (MYB) gene alone . Purple color also occurs in leaves, leaf sheaths, internodes, and ligules. A genic model suggested that coloration depended on three different kinds of genes, namely a color-producing gene, an activator gene for anthocyanin biosynthesis, and a tissue-specific pigmentation gene  with functional Rd (dihydroflavonol reductase), whereas brown pericarp is formed without Rd participation  has various colors such as black hull, red pericarp, purple awn, and purple leaf margin  for constructing mapping populations were as follows:2F6 generation in Nipponbare background. The donor parent was a temperate japonica variety, XZM, with purple hulls.(i) PH NIL, a purple hulled NIL of BC4F8 generation. It was obtained from the crosses and backcrosses between Nipponbare and a temperate japonica with purple awns.(ii) PA NIL, a purple awned NIL of BCNILs for transformation, expression and/or metabolite detection studies were as follows:4 population I-2. It has straw-white hulls caused by non-functional C1 and A1 alleles.HC1, a cSa-type NIL selected from recessive plants in F4 population I-1. It has purple hulls caused by functional C1, S1, and A1 alleles.HC2, a CSA-type NIL selected from the dominant segregates in F4-III. It has brown hulls caused by functional C1 and S1 with non-functional A1 alleles.HC3, a CSa-type NIL derived from recessive segregates of the line F4 population Ⅰ-1. It has straw-white hulls caused by functional S1 and A1 with non-functional C1 alleles.HC4, a cSA-type NIL selected from the recessive segregates in F3F6 generation selected from recessive plants of the line F4-II. It has purple apiculus caused by functional C1 and A1 with non-functional S1 allele.HC5, a CsA-type NIL with BCC1 and A1 of 108 wild rice varieties were supplied by Q. W. Yang.Cultivars used for haplotype analysis, neutrality tests and phylogenetic analyses were collected from the 3K-rice project . The genome sequences of All rice plants were grown under natural conditions in paddy ﬁelds located in Beijing or Sanya in Hainan province. Detailed information for accessions is provided in C1 (Os06g0205100) from genomic DNA and the S1 (Os04g0557500) coding DNA sequence (CDS) from cDNA of PH NIL; the PCR products were digested with BamHI and SpeI, followed by cloning into the binary vector pMDC32  of C1 was amplified from DNA of PH NIL, with digestion by PmeI and AscI, and cloned into the pMDC83 vector , 0.2 μl Rox Reference Dye II (Takara), 0.4 mΜ gene-specific primers and 0.5 μl cDNA on an ABI 7500 real time PCR system (Applied Biosystems). The ubiquitin gene Os03g0234200 was used as an internal reference.Total RNA was extracted from various rice tissues using RNAiso Plus (Takara). cDNA was generated in 25 μl reaction mixtures containing 2 μg DNase I-treated RNA, 200 U M-MLV reverse transcriptase (Takara), 40 U Recombinant RNase Inhibitor (Takara) and 0.1 μΜ oligo (dT)C1 was amplified and subcloned into the pGADT7 vector (Takara) between EcoRI and BamHI sites. Intact S1 or its truncated CDS was fused to the pGBKT7 vector between the EcoRI and BamHI sites. Transformed yeast cells were grown on SD−Trp/−Leu (−LT) or SD−Trp/−Leu/−His/−Ade(−LTHA) media plus X-gal. Experimental procedures were performed according to the manufacturer’s user manual (Clontech). Yeast strain AH109 was used in this assay.Intact or a series of truncated CDSs of CHS (Os11g0530600), CHI (Os03g0819600), F3H (Os04g0662600), F3′H (Os10g0320100), ANS (Os01g0372500), and A1 were cloned and fused to the 5′ end of the β-glucuronidase (GUS) gene in the pMDC163 vector as reporters . DNA probes were amplified and labeled with biotin (Invitrogen/Thermo Fisher Scientific). DNA gel shift assays were conducted according to the manual for the LightShift Chemiluminescent EMSA Kit .The DNA binding domain of C1 or S1 was amplified from PH-NIL and cloned into the pMAL-C5X vector  using the Seamless Assembly Cloning Kit . MBP and MBP–C1−1 lidocaine . Samples were shaken three times during this period to improve extraction efficiency. After centrifugation for 10 min at 10 000 g, the supernatant was filtered through a 0.22 μm syringe filter and then stored at −80 °C. Each colored hull was detected and assayed in three replications.Fully mature seeds were harvested from the field and dehulled to collect the hull samples. Each colored hull sample was derived from five individual plants and mixed equally. The vacuum freeze-dried hulls were ground using a mixer mill  for 1.5 min at 30 Hz; 100 mg powdered hull was extracted overnight at 4 °C with 1.0 ml 70% aqueous methanol containing 0.1 mg l−1; temperature, 40 °C; injection volume, 5 μl. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)–MS. Quantiﬁcation of metabolites was carried out using a scheduled multiple reaction monitoring method –electrospray ionization (ESI)–tandem mass spectrometry (MS/MS) system . Analytical conditions were as follows, HPLC: column, Waters ACQUITY UPLC HSS T3 C18 ; solvent system, water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program, 100:0 v/v at 0 min, 5:95 v/v at 11.0 min, 5:95 v/v at 12.0 min, 95:5 v/v at 12.1 min, 95:5 v/v at 15.0 min; flow rate, 0.4 ml minwww.r-project.org/) to visibly display the specific accumulation of flavonoids and variations among purple, brown, and straw-white hulls.Relative signal intensities of metabolites were normalized by ﬁrst dividing them by the intensities of the internal standard (lidocaine) and then log2 transforming to improve normality. Principal component analysis was carried out by SIMCA-P software. Differences in the metabolites between three types of colored hulls were determined by two criteria: (i) variable influence on projection (VIP) values obtained from the orthogonal partial least squares discrimination analysis (OPLS-DA) model (VIP≥1), and (ii) fold change ≥2 or fold change ≤0.5 between pairwise comparisons. After conditional selection, 49 flavonoids were used for hierarchical clustering analysis by R  of software . The Arlsoftware .japonica, 228 indica) accessions was used to evaluate the π and Tajima’s D values of the two genes. For C1, single nucleotide polymorphisms (SNPs) in a 5 kb (3 kb upstream and 1 kb downstream of the C1) region were used for analysis; for A1, SNPs in a 6 kb region covered by A1 were selected. The data were computed by DnaSP 5.10 software  were surveyed for the C1 and A1 in wild rice, haplotypes containing only one variety were also generated in a spanning tree. But for simplification, we only chose haplotypes containing more than two individuals or integrated haplotypes of single individuals located in the main branch as representative haplotypes. Finally, 75 wild rice, 199 indica and 127 japonica accessions were selected to construct a minimum-spanning tree. A total of 76 polymorphic sites including 22 (18 SNPs and four indels) from the C1 gene region and 54 (53 SNPs and one indel) from the A1 gene region were used in the study. The combined C1 and A1 haplotypes of 401 varieties were genotyped by MEGA 6 software and used for constructing a neighbor-joining tree. The data were also used to generate a minimum spanning tree as mentioned above.Because of the large sequence diversities for 1 had purple hulls indicating dominance of this trait . A further backcross was made to Nipponbare to study the inheritance of hull coloration. The Fis trait . The F2 ite hull . Under fite hull . Purple the hull .4 populations of F3 individuals that had colored phenotype. Among these lines, we identified several populations segregating in a single gene and proposed a genetic model for hull pigmentation. The inheritance patterns of I-1, -2, -3, and -4 indicated there is a color-producing (chromogen) gene because the recessive classes had straw-white hull phenotypes. Line F4-II segregated purple hull and purple apiculus in a 3:1 ratio suggesting that the locus involved was specific for hull pigmentation. Finally, the locus responsible for purple versus brown in line F4-III was likely an activator of anthocyanin biosynthesis  to be the candidate gene for chromogen.Firstly, we used recessive individuals obtained from Fme 6 see . Within  pathway . Thus, w4-II and its progeny. The candidate gene was firstly mapped to a 345 kb region between SSR markers RM3820 and MM2687 on chromosome 4. Due to lack of polymorphic markers between the PH NIL and Nipponbare, we generated another mapping population using the PA NIL (straw-white hull with purple awn) crossed with the PH NIL  indicating that a single dominant gene from the PH NIL controlled hull pigmentation. This population was used for fine-mapping of the causal gene. Finally, we mapped the candidate gene to a 50.02 kb interval between SNP markers Snp2 and Snp4. The single gene in this region, Os04g0557500, encodes a bHLH transcription factor  were screened by SSR markers, and finally the candidate gene was mapped to a 49 kb region on chromosome 1. In the mapped region, Os01g0633500 encodes a dihydroflavonol reductase (DFR) predicted to catalyse the conversion of dihydroflavonols to leucoanthocyanidins, a crucial step in the biosynthesis of anthocyanin . All three genes were functional for hull pigmentation in PH NIL, but not in Nipponbare.Ftion see . We nameC1 allele from PH NIL under its native promoter or the cauliflower mosaic virus (CaMV) 35S promoter was transformed into Nipponbare. Both the complementation and overexpression transgenic lines produced brown colors, but it appeared only in the apiculi  was significantly decreased relative to those in HC2. We therefore speculate that C1 plays a more important role than S1 in activating expression of structural genes in the hulls.We next investigated the expression patterns of the structural genes under C1 and/or S1 regulation in the three NILs, HC2, HC4 and HC5. As shown in trans-activation assays in Nicotiana benthamiana. GUS/LUC ratios indicated that neither C1 nor S1 could separately activate expression of structural genes. However, when C1 and S1 were both expressed in tobacco leaves, GUS activities were strikingly elevated in all six kinds of transformants , HC3 (brown), and HC4 (straw-white). A total of 161 flavonoids were detected see . PrincipBy pairwise comparisons of the flavonoid metabolite contents in the three types of hulls, we found the most abundant types of flavonoids were highly accumulated in purple hulls and the lowest levels were in straw-white hulls . Among tO-glucoside (C3G)  and nine non-functional haplotypes (hap5–13). In the non-functional alleles, three kinds of indels were identified in which 10 bp deletion occurred in almost all indica varieties, whereas −TC and −GAG deletions mainly occurred in temperate japonica accessions. Besides, a 45 bp substitution between positions 493 and 537 occurred only in temperate japonica accessions . All of these variations implied that non-function mutations in C1 arose independently in indica and japonica. Among A1 haplotypes, there were nine functional (hap1–9) and three non-functional types (hap10–12). All three non-functional alleles were only in temperate japonica, two (hap11 and hap12) of which were newly found as rare variations  in 471 varieties were surveyed. Nonsense mutations were not found in any of the five genes  whereas straw-white cultivars had been subject to directional selection   see . Tests oarieties . In brieC1 and A1 in a panel of 342 accessions. Compared with landrace groups, the functional allele frequencies of C1 in the improved rice groups were significantly reduced in both indica and japonica. There was a similar trend for A1 in japonica  and their supposed wild rice ancestors were from Southeast Asia and South China. We assumed that geographically distributed indica varieties independently originated from wild rice in their local areas . Although pigmentation depends on a series of genes belonging to the flavonoid pathway, natural variations that caused functional mutations mainly occurred in these three genes. It seems that this gene system is universal in regulating various organs’ pigmentation. Nonetheless, we cannot exclude the possibility that other R2R3-MYB or bHLH proteins participate in the regulatory network. We collected seven black rice varieties and all had the functional A1 allele. Three of these varieties had functional C1 and also possessed purple color in the hull. But in the other four varieties, purple color appeared only in the pericarp, and C1 in these four varieties showed loss-of-function . This indicates that Os02g0682500 may not be essential for anthocyanin biosynthesis in rice. It could also be that this gene plays an important role in rice growth and any functional mutations in this gene may have a lethal effect. Besides, it also should be noted that the anthocyanin-related WD40 proteins are functionally redundant in rice. More work is needed to clarify this.The MBW complex consists of MYB–bHLH–WD40 transcription factors and is the main regulatory unit for anthocyanin/proanthocyanidins biosynthesis in plants (ZmA1 (A1 homolog in maize) by either protein directly binding to a specific DNA sequence in the ZmA1 promoter. The DNA binding motifs of C1 and S1 in the promoter of A1 are the same as that in the maize ZmA1 promoter  interacts with the N-terminal region of ZmB/R (S1 homologs in maize) . The comOryza sativa, Zea mays, and Arabidopsis, we found that this MYB transcription factor in the three species had higher similarity in the R2 and R3 domains (86%) when compared to other regions (31%) (data not shown). Likewise, S1, ZmB/R, and TT8, as homologs in the three species, had a higher similarity in their MYC N-terminal regions (74%) than their C-terminal regions (data not shown). We consider that these conserved domains are fundamental in maintaining their functions in regulating flavonoid biosynthesis in plants.By homologous comparisons of C1 protein sequences between In wild rice, purple color occurs in various organs, but not in the hull and pericarp . The pigS1 acted as a hull-specific pigmentation gene, but the functional nucleotides were not identified. We found that the promoter sequence of S1 in the PH NIL was the same as that in Nipponbare, but different changes may occur in the 3′ end of S1 in PH NIL. We failed to amplify products from the seventh exon to the 3′-UTR in PH NIL. However, amplification succeeded in PA NIL and its sequence was same as that in Nipponbare. But further investigation of the S1 sequence in 13 varieties with purple hull revealed that the 3′-end ‘black hole’ was not universal (see S1 rather than mutations in translated products have a determinant effect on the acquisition of pigmentation in the hull.In the present study, we confirmed that rsal see . We specSupplementary data are available at JXB online.Fig. S1. Color phenotypes in rice floral organs.Fig. S2. Plasmid constructs for the transient expression assay.C1.Fig. S3. Mapping of S1.Fig. S4. Mapping of A1.Fig. S5. Mapping of C1 in overexpressed transgenic lines.Fig. S6. Expression of C1 and A1 in C1 RNAi lines.Fig. S7. Expression of S1 in PA NIL.Fig. S8. Overexpression of Fig. S9. Principal component analysis of the LC-MS data.C1 and A1.Fig. S10. Haplotype analyses of Oryza sativa.Fig. S11. Proposed evolutionary pathway of C–S–A gene system manipulating rice coloration.Fig. S12. Proposed model of S1 in NIP.Fig. S13. Overexpression of S1 in 13 purple hulled accessions by PCR.Fig. S14. Investigation of the 3′ end structure of Table S1. Information on rice varieties used in this study.Table S2. Primers used for fine mapping, gene sequencing, RT-PCR, and vector construction.C1 and A1 haplotypes in accessions from the rice mini core collection.Table S3. Summary of the Table S4. Summary of phenotypic data of segregating lines.Table S5. Data matrix of 161 flavonoid metabolites detected in rice hulls.Table S6. Data sets of differentiated metabolites between pairwise comparisons of three groups.Table S7. Forty-nine flavonoid metabolites with differences among lines with purple, brown, and straw-white hulls.Table S8. Major flavonoid compounds identified in purple and brown hulls.Table S9. Natural variations in CDS regions of five structural genes.C1.Table S10. Nucleotide variation and neutrality test of A1.Table S11. Nucleotide variation and neutrality test of Table S12. Seven rice varieties with purple pericarp in the mini core collection.Supplementary Figures and TablesClick here for additional data file.Supplementary Table 1Click here for additional data file.Supplementary Table 2Click here for additional data file.Supplementary Table 3Click here for additional data file.Supplementary Table 5Click here for additional data file.Supplementary Table 6Click here for additional data file.Supplementary Table 7Click here for additional data file.


Four patients (2 newly diagnosed patients and 2 with relapsed MM) were included and underwent whole-body PET/CT after injection of [18F]FPRGD2. The obtained images were compared with results of low dose CT and already available results of a combined [18F]NaF/[18F]FDG PET/CT. In total, 81 focal lesions (FLs) were detected with PET/CT and an underlying bone destruction or fracture was seen in 72 (89%) or 8 (10%) FLs, respectively. Fewer FLs (54%) were detected by [18F]FPRGD2 PET/CT compared to low dose CT (98%) or [18F]NaF/[18F]FDG PET/CT (70%) and all FLs detected with [18F]FPRGD2 PET were associated with an underlying bone lesion. In one newly diagnosed patient, more [18F]FPRGD2 positive lesions were seen than [18F]NaF/[18F]FDG positive lesions. This study suggests that [18F]FPRGD2 PET/CT might be less useful for the detection of myeloma lesions in patients with advanced disease as all FLs with [18F]FPRGD2 uptake were already detected with CT alone.An observational study was set up to assess the feasibility of [ The Rns NaF and [18F]FDG for PET/CT is another strategy to improve the detection of bone metastases and was first introduced by Iagaru et al. [18F]NaF, allowing the detection of bone metastases with bone formation, and [18F]FDG, enabling the detection of metastases with increased rate of glucose metabolism, was to improve the sensitivity for detecting metastatic lesions. A prospective clinical trial evaluating combined [18F]NaF and [18F]FDG for PET/CT in patients with MM is currently under investigation (EudraCT 2013-004807-38), aiming at comparing its capacity to detect MM lesions with the capacities of magnetic resonance imaging, CT alone, and whole-body X-rays [The combination of FNaF and u et al. . The rat18F]FPRGD2 PET/CT to identify myeloma lesions. Secondarily, the detection rate of [18F]FPRGD2 PET/CT was compared to CT alone. Additionally, [18F]FPRGD2 PET/CT images were compared to combined [18F]NaF/[18F]FDG PET/CT images, available for those patients that were also included in the above-mentioned trial [The current observational study was set up to assess the feasibility of [ed trial .Patients with newly diagnosed or relapsed MM were prospectively included. This study was registered as EudraCT #2013-004807-38 and was approved by the Ethics Committee of the academic hospital (CHU of Liege). All subjects provided written informed consent for this study.18F]FPRGD2 was performed as previously reported and in compliance with current good manufacturing practice regulations FPRGD2 PET/CT and combined [18F]NaF/[18F]FDG PET/CT . PET/CT scans were acquired in a Gemini TF scanner after injection of 296 ± 9 MBq [18F]FPRGD2 (median uptake time: 62 min) or 133 ± 6 MBq [18F]NaF and 242 ± 27 MBq [18F]FDG (median delay between [18F]FDG and [18F]NaF injections: 2 min and uptake time: 66 min). All patients fasted for 6 h prior to radiopharmaceutical injection . A low dose CT  followed by the PET emission scan of 90 seconds per bed position was performed.Every patient underwent whole-body (WB) scans, from vertex to toes, using NaF/[18F]FDG PET in the femur did not show any abnormality on CT images. Overall, the detection rate of [18F]FPRGD2 PET was lower than [18F]NaF/[18F]FDG PET, whatever the FL location, and the mean uptake (SUVmax) of [18F]FPRGD2 was overall lower than [18F]NaF/[18F]FDG  showed [18F]NaF/[18F]FDG positive lesions without [18F]FPRGD2 uptake.Four patients with MM were included, apsed MM . Based o[18F]FDG . Out of 2 uptake . NonetheG uptake . In patiG uptake , both FDG PET . In pati]FDG PET , [18F]FP18F]FPRGD2 PET/CT and to assess its feasibility in MM disease. In the studied patients, the detection rate of [18F]FPRGD2 PET was lower than the detection rate of low dose CT alone  were not studied and could be of interest. On the other hand, the high bone marrow background activity related to [18F]NaF uptake may explain why some of the FLs detected with [18F]FPRGD2 PET/CT were not seen with [18F]NaF/[18F]FDG. Diffuse bone marrow infiltration was not reliably estimated with [18F]NaF/[18F]FDG PET due to high [18F]NaF bone uptake while it was suspected with [18F]FPRGD2 PET/CT in 2 of the 4 patients , tumor cells, and activated osteoclasts, while [18F]NaF/[18F]FDG uptake reflects tumor cell metabolism and/or bone formation. The heterogeneous uptake of [18F]FPRGD2 can be explained by biological phenomena and previously received treatments. The myeloma-induced angiogenesis appears after an “angiogenic switch” due to the release of angiogenic factors by subsets of myeloma cells or can be directly in proportion to the tumor infiltration inside the bone marrow [18F]FDG was recently found to be associated with reduced expression of hexokinase-2, responsible for the first step of glycolysis FPRGD2 PET/CT might not be appropriate for detection of MM lesions, it may be of use in the assessment of integrin αvβ3 expression in MM lesions, especially in clinical trials evaluating inhibitors targeting αvβ3 integrins, as recently investigated by Tucci et al. [18F]FPRGD2 PET/CT might be useful to detect bone marrow infiltration in precursor states of the disease .Even though this case report suggests that [i et al. . In addi18F]FPRGD2 PET/CT detected only 50% of the FLs detected by CT suggesting that the clinical utility of [18F]FPRGD2 PET/CT is rather limited for the detection of overt MM lesions. However, the clinical and possibly prognostic relevance of [18F]FPRGD2 positive MM lesions needs further investigation.In this case report, [


F8-deficiency on VWF plasma levels in F8-/y mice is unresolved. In order to avoid confounding variables, we back-crossed F8-deficient mice onto a pure C57BL/6J background and analysed VWF plasma concentrations relative to C57BL/6J WT (F8+/y) littermate controls. F8-/y mice showed strongly elevated VWF plasma concentrations and signs of hepatic inflammation, as indicated by increased TNF-α, CD45, and TLR4 transcripts and by elevated macrophage counts in the liver. Furthermore, immunohistochemistry showed that expression of VWF antigen was significantly enhanced in the hepatic endothelium of F8-/y mice, most likely resulting from increased macrophage recruitment. There were no signs of liver damage, as judged by glutamate-pyruvate-transaminase (GPT) and glutamate-oxalacetate-transaminase (GOT) in the plasma and no signs of systemic inflammation, as white blood cell subsets were unchanged. As expected, impaired haemostasis was reflected by joint bleeding, prolonged in vitro clotting time and decreased platelet-dependent thrombin generation. Our results point towards a novel role of FVIII, synthesized by the liver endothelium, in the control of hepatic low-grade inflammation and VWF plasma levels.Von Willebrand factor (VWF) is the carrier protein of the anti-haemophilic Factor VIII (FVIII) in plasma. It has been reported that the infusion of FVIII concentrate in haemophilia A patients results in lowered VWF plasma levels. However, the impact of  Haemophilia A is a rare X-linked recessive bleeding disorder that is caused by the deficiency or absence of FVIII . In abouF8 gene for our study [F8-/- mouse line onto a pure C57BL/6J genetic background using a speed congenics approach. While in haemophiliacs spontaneous bleeding is frequently observed in the joints (haemarthrosis) and in the musculature (haematomas), F8-deficient mice do not show spontaneous bleeding episodes. Nevertheless, well-defined bleeding models, such as the needle-induced knee bleeding model, have been developed to study haemophilia and anti-haemophilic therapies in mice [We have used the haemophilia A mouse model with disrupted exon 16 of the ur study . As VWF ur study , we backin vivo is activated through limited proteolysis by thrombin and binds FIXa to accelerate the formation of FXa in the so-called Xase complex, thus amplifying thrombin generation yielding in quantitative formation of fibrin [in vitro analyses and the wealth of studies on haemophilia A, the significance of genetic F8-deficiency, as it occurs in haemophilia A, on VWF plasma levels and immune cell infiltration of the liver is unexplored.During coagulation, FVIII f fibrin . The antf fibrin . By tissf fibrin , 9. As af fibrin –12. Whilf fibrin , 14, thef fibrin –17. In sex vivo, and to determine its implication in hepatic inflammation, we have used the F8-deficient haemophilia A mouse model [F8-deficient mice revealed elevated VWF content in the liver endothelium along with signs of low-grade hepatic inflammation and increased VWF plasma levels compared with their co-housed WT littermate controls.To systematically investigate the role of FVIII on VWF content in the liver endothelium, on VWF plasma levels, and VWF multimer size se model  on a purtm1Kaz/J were purchased from Jackson Laboratory . The B6;129S4-F8tm1Kaz/J were backcrossed according to marker assisted selection breeding (speed congenic) to the C57BL/6J background by Taconic biosciences . Only male C57BL/6J WT and C57BL/6J F8-/y or C57BL/6J F8+/y mice from heterozygous breedings were used for this study. All experimental animals were 8–14 weeks old and housed in conventional cages (max 5 mice per cage) with a 12h light-dark cycle. All experiments performed in mice were approved by the local committee on legislation on protection of animals C57BL/6J WT and B6;129S4-F8Whole blood was collected by cardiac puncture. For differential blood analysis blood was transferred to EDTA tubes, for GOT and GPT measurements heparinized tubes were used. Both analyses were performed by the Insitute of Clinical Chemistry and Laboratory Medicine at University Medical Center Mainz.Mice were injected subcutaneously with Buprenorphine (0.1mg/kg BW)  30 min prior to the experiment. They were anesthetized intraperitoneally with midazolam (5 mg/kg BW)  and medetomidine (0.5 mg/kg BW) . The fur was removed from both legs. The external joint diameter was calculated as the mean of three independent measurements per leg by a Mitutoyo thickness measuring system . Joint bleeding was induced to the right knee by transcutaneous insertion of a 30 G needle through the patellar ligament and into the infrapatellar fat pad, avoiding contact with the cartilage and bone. Subsequently, the needle was withdrawn gently. Left knee served as an intra-animal control. 0.1 mg/kg BW Buprenorphine was administrated subcutaneously at 6 h after injury and was continued 3- times per day, for the following 2 days. On the second experimental day, mice were anesthetized as described above and the weight and the external joint diameter were determined. Directly thereafter the mice were euthanized.2+ directly prior to measurement . Reconstitution of the FVIII activity was succeeded by adding 2.5 U/ml human recombinant FVIII. Clotting time and clot formation time were measured by a whole blood haemostasis analyzer .Citrated (3.8%) whole blood was collected by cardiac puncture. Blood was recalcified with 20 mM Ca3, 2.7 mM KCl, 0.42mM Na H2PO4, 5 mM Hepes, pH 7.4) and centrifuged at 100 x g for 4 min at room temperature to obtain platelet-rich plasma (PRP). Platelet-poor plasma (PPP) was prepared by centrifugation of residual PRP including the buffy coat at 30’000 x g for 10 min at room temperature. Thrombin generation was determined in PRP triggered by tissue factor  or by α-thrombin  (0.1 U/mL) and in PPP triggered by tissue factor in the presence of phospholipids  with fluorogenic calibrated automated thrombography, FluCa-Kit .Citrated blood was collected by cardiac puncture, diluted with one volume Tyrode’s buffer  according to manufacturer’s instructions and was then reverse transcribed . Total RNA (2 μg) was reverse transcribed with the High Capacity cDNA Reverse Transcription Kit  and SYBR green-based qRT–PCR was performed with iQ SYBR Green Supermix . The oligonucleotide sequences used are listed in ®-Histofix 4%  and provided to the Core Facility Histology of the University Medical Center Mainz for F4/80 and VWF staining . Paraffin sections were routinely stained with haematoxylin eosin. All sections were analyzed by light microscopy  and scored in a blinded fashion by three different experts.Tissues were fixed in RotiF8-/y mice. The F8+/y remained untreated. Whole blood was collected from the orbital sinus in EDTA tubes 2h after the injections and centrifuged at 800 x g for 10 min to aquire PRP. For platelet poor plasma (PPP), PRP was centrifuged at 1.200 x g for 10 min.50 U/kg BW of recombinant FVIII  or equal volume of 0.9% NaCl were injected into the lateral tail vein of 2SO4. The absorbance was measured at 450nm by an Opsys MR microplate reader .A 96 well plate was coated overnight with anti-human VWF antibody  and was then blocked for 1 hour with 1% BSA in PBS pH 7.4. Blood was collected from the facialis vein in a K2 EDTA microvette tube  and PPP was prepared by centrifugation at 800 x g for 10 min followed by centrifugation at 1.200 x g for 10 min. A standard curve was created from increasing dilutions of pooled human EDTA plasma with defined VWF concentration obtained from 10 healthy blood donors. PPP and standards were incubated for 1 h followed by 3 washing steps with 0.2% Tween in PBS. The polyclonal rabbit anti-human VWF/HRP was incubated for 1 h  and the plate was washed 3 times. 80 μl of the chromogenic substrate TMB (Sigma) were added and the reaction was terminated by adding 80 μl 2N H™ BCIP/NBT, Sigma-Aldrich).Frozen citrated plasma samples were thawed in a 37°C water bath for 10 min and gently mixed. Thawed plasma samples (30 μL) were diluted in 30 μl of 2x sample buffer , 130 μl of 1x sample buffer  and supplemented with 10 μl of 0.2% solution of sodium bromphenol blue. They were incubated 30 min at 58°C in a water bath and centrifuged for 1 min at full speed. Electrophoresis samples were maintained at RT until loading. SeaKam HGT (P) high gelling temperature agarose  was used to prepare a stacking gel  and 2-Hydroxyethyl agarose  was used for the separating gel . A horizontal slab gel electrophoresis unit  was water-cooled to 6°C prior to and throughout electrophoresis. Agarose gels were placed on the horizontal ceramic electrophoresis cooling slab. Filter paper wicks, immersed in tanks containing electrophoresis running buffer  were placed on the cathode (sample) and anode ends of each agarose slab. Plasma samples (45 μl) were loaded on the agarose gel sample wells. Electrophoresis began at constant current (13 mA) for approximately 2 h to move the plasma proteins into the stacking gel. The wells were then sealed with additional stacking gel and electrophoresis resumed at constant current of 15 mA and 1–3 W overnight, until termination of electrophoresis when the dye front reached the anodal wick. After stopping electrophoresis (day 2), gels were incubated for 15 min in electrophoresis running buffer before transfer to nitrocellulose membrane by wet tank blotting for 3 h at constant current (1 A) in blotting buffer (0.008 mol/L Na2HPO4). The membrane was blocked for 1 h with blocking buffer  at RT and incubated in rabbit anti-human VWF  over night at 4°C. After 3x washing (0.1% Tween in TBS) membrane was incubated with AP-conjugated goat anti-rabbit antibody  for 2 h at RT. After 3x washing, separated multimers were visualized using a buffered solution containing BCIP/NBT, pH 9.5 (SIGMA FASTT = 20-RFUT = 0). A standard curve was constructed by plotting the ΔRFU for each standar4d versus the corresponding concentration.The ADAMTS-13 activity was measured by the Actifluor ADAMTS-13 activity assay according to manufacturer’s instructions . Briefly, citrated blood was collected by cardiac puncture and platelet poor plasma was prepared as described previously. Assay buffer (provided) was added in each well of a 96 well plate followed by samples and standards (provided). The plate was warmed for 3 min before addition of the ALEXA488-vWF86 FRET substrate. The plate was read with excitation at 485 nm, emission at 535nm and cut off filter at 530 nm by a fluoroskan ascent FL . Afterwards, it was incubated for 20 min at 37°C and read once more with the same settings. The net charge in fluorescence was calculated (ΔRFU) as RFUSialic acid ELISA was purchased from MyBioSource  and performed according to manufacturer’s instructions with undiluted EDTA plasma samples.F8-/y mice were injected twice on alternate days for 4 days with 3F-NeuAc (Inhibitor) at a dosage of 100 mg/kg BW in the lateral tail vein. WT animals in the control group received 20 μl DMSO solved in NaCl to a volume of 100 μl following the same injection protocol. Afterwards whole blood was collected from the orbital sinus, transferred to EDTA tubes and platelet poor plasma was prepared as described previously. PPP was analyzed by VWF and sialic acid ELISA kit.Sialylation inhibitor 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-NeuAc) was purchased from Merck Millipore . The inhibitor was dissolved in DMSO and diluted 1:5 in NaCl to a volume of 100 μl. t-test, one-way analysis of variance (ANOVA), or two-way ANOVA. * p< 0,05, ** p<0,01, ***p<0,001.All data and statistical analysis were performed using GraphPad Prism 6 for Windows . Data are presented as mean values ± standard error of the mean. Statistical analyses were performed using the student’s unpaired F8-deficient mouse [F8+/y (WT) and F8-/y (F8-deficient) mice (F8-deficient mice showed a 20% increase in joint swelling diameter compared with littermate WT controls at 24 hours after puncture (F8-/y) was characterized by significantly prolonged clotting time (CT) as well as increased clot formation time (CFT)  at a concentration of 2.5 U/ml (F8-/y whole blood was 2 to 3 times prolonged compared with F8+/y controls (F8-deficient mice on a C57BL/6J genetic background display the expected bleeding phenotype.The nt mouse  is a gennt) mice , excludipuncture . In throme (CFT) . The mea2.5 U/ml . Also thcontrols . Our resF8-/y mice compared to F8+/y controls (F8-deficiency resulted in nearly complete loss of thrombin generation not only in platelet-rich plasma (PRP), but also when either 1 pM tissue factor (TF) or 0.1 U/ml thrombin (thr) was used as a trigger. The thrombin peak and the endogenous thrombin potential (etp) were strongly reduced in controls . CollectF8-deficiency in highly vascularized organs, we analyzed liver and lung tissues. As expected, FVIII mRNA expression was strongly decreased in the liver of F8-/y mice relative to F8+/y controls  at a dose of 1.5 U per 30 g body weight, or with the same volume of 0.9% NaCl solution as a vehicle control. While F8-/y mice again showed increased VWF plasma levels relative to the untreated F8+/y controls, we found that substitution of FVIII diminished VWF plasma levels  in murine whole blood. This is due to the crucial role of FVIII in platelet-dependent thrombin generation, when low to moderate concentrations of tissue factor or thrombin are used as a trigger.The most common complication of haemophilia A is recurrent haemarthrosis in the knee joints that leads to chronic degenerative arthropathy . In the ous work , we recoF8-deficient mice does not significantly differ from WT littermate control platelets [F8-/y mice, induced by low to moderate concentrations of TF or thrombin.With washed platelets, it has been demonstrated that the activation state of platelets from latelets . Howeverlatelets . This isF8-deficient genetic mouse model revealed increased VWF content in the liver endothelium together with increased VWF plasma levels, which was associated with hepatic low-grade inflammation. We found elevated TNF-α, CD45, and TLR4 mRNA levels in the livers of F8-/y mice that are hallmarks of inflammation in the liver. Moreover, increased F4/80 macrophage counts and increased F4/80 transcript levels were detected in the livers of F8-/y mice, indicating that F8-deficiency results in a chronic inflammatory liver phenotype. Low-grade inflammation could potentially explain the increased VWF content detected in the liver endothelial cells.Most interestingly, the back-crossed littermate F8 deficiency leads to increased plasmatic VWF levels in mice is in line with the published report that haemophilia A patients that were injected with FVIII concentrate showed reduced VWF plasma levels [F8-/y mice with recombinant human FVIII led to lowering of VWF plasma levels. In contrast to the biochemical in vitro studies that have demonstrated a synergistic role of FVIII and platelets on the cleavage of VWF multimers by ADAMTS13 [F8-deficient C57BL/6J mouse line were not associated with altered VWF multimer patterns.Our finding that a levels . Also, tADAMTS13 –17, the in vivo inhibition of sialylation with the global sialyltransferase inhibitor 3F-NeuAc reduced VWF antigen levels in the plasma of F8-/y mice. However, since we did not detect differences in the overall sialylation of plasma proteins comparing F8+/y WT mice with F8-/y mice, the unchanged multimer pattern along with increased VWF plasma levels could not be explained by the increased hepatic expression levels of the modifier gene ST3Gal4 in F8-/y mice [F8-/y mice. Based on the elevated inflammatory markers in F8-/y mice, it will be interesting to pinpoint the role of FVIII in the regulation of low-grade inflammation of the liver. In contrast to clinical hemophilia A studies, which due to patient heterogeneity and ongoing substitution therapy are prone to confounding factors, the established mouse model could be instrumental to study effects of F8-deficiency on inflammatory phenotypes and associated co-morbidities of the disease.Our results clearly demonstrate that -/y mice . TherefoS1 FigA) White blood cell counts (n = 5). (B) red blood cell counts (n = 5). (C) hemoglobin levels (n = 5). (D) platelet counts (n = 5). (E) Neutrophil counts (n = 5). (F) Lymphocyte counts (n = 5). (G) Monocyte counts (n = 5). (H) Eosinophil counts (n = 5). (I) Basophil counts (n = 5). All data were expressed as means ± SEM. Statistical comparisons were performed using the Student’s t-test.((TIF)Click here for additional data file.S2 FigA) CLEC4M , (B) ASGR1 , (C) ASGR2 (n = 7), (D) SiglecF , (E) ST3Gal4 . Lung transcript levels of (F) LRP1 (n = 6), (G) ST3Gal4  and (H) VWF (n = 7). All data were expressed as means ± SEM. Statistical comparisons were performed using the Student’s t-test, * p< 0.05.Hepatic transcript levels of ((TIF)Click here for additional data file.S3 FigA) VWF plasma levels and (B) sialic acid plasma levels of untreated F8+/y (WT)  and F8-/y mice  vs WT intravenously injected with vehicle control (DMSO) (n = 6) and F8-/y mice i.v. treated with 100mg/kg body weight 3F-NeuAc (Inhibitor) (n = 4), respectively. All data were expressed as means ± SEM. Statistical comparisons were performed using one-way ANOVA, * p< 0.05, ** p<0.01.((TIF)Click here for additional data file.


The cellular and molecular mechanisms underpinning the unusually high virulence of highly pathogenic avian influenza H5N1 viruses in mammalian species remains unknown. Here, we investigated if the cell tropism of H5N1 virus is a determinant of enhanced virulence in mammalian species. We engineered H5N1 viruses with restricted cell tropism through the exploitation of cell type-specific microRNA expression by incorporating microRNA target sites into the viral genome. Restriction of H5N1 replication in endothelial cells via miR-126 ameliorated disease symptoms, prevented systemic viral spread and limited mortality, despite showing similar levels of peak viral replication in the lungs as compared to control virus-infected mice. Similarly, restriction of H5N1 replication in endothelial cells resulted in ameliorated disease symptoms and decreased viral spread in ferrets. Our studies demonstrate that H5N1 infection of endothelial cells results in excessive production of cytokines and reduces endothelial barrier integrity in the lungs, which culminates in vascular leakage and viral pneumonia. Importantly, our studies suggest a need for a combinational therapy that targets viral components, suppresses host immune responses, and improves endothelial barrier integrity for the treatment of highly pathogenic H5N1 virus infections. In healthy individuals, the symptoms of seasonal influenza virus infection are mild and the infection is cleared within 4–7 days. However, infection with highly pathogenic avian influenza virus (H5N1) can be severe and often results in fatal pneumonia even in healthy adults. While it is known that both viral and host factors play a role in enhanced disease progression, the molecular mechanisms for the high virulence of H5N1 virus are not completely understood. In this study, we engineered avian influenza H5N1 viruses incapable of replicating in endothelial cells and evaluated disease symptoms in mice and ferrets. Our studies show that H5N1 infection of endothelial cells causes severe disease and death of infected animals in part due to the damage of endothelial cells lining the blood vessels, which results in leakage of fluid into the lungs (pneumonia). Orthomyxoviridae family, pose a constant threat to human health with seasonal epidemics and occasional pandemics. It is estimated that seasonal influenza virus infections result in 250,000–500,000 annual deaths worldwide ..7 plaqueTo determine if endothelial cell or hematopoietic cell tropism contributes to virulence, C57BL/6J mice were intranasally infected with different doses  of the H5N1 viruses, and monitored for weight loss and survival for 14 days. Mice infected with H5N1-ScrbT showed pronounced weight loss (>25%) and the majority of mice succumbed to infection at both the 10 and 25 PFU dose . In contA prior study using WSN/33 (a mouse adapted strain) indicated that endothelial cells contribute to excessive production of inflammatory cytokines . In addi- CD31+) in H5N1-ScrbT and H5N1-142T infected mice as compared to H5N1-126T infected mice (+) was lower in H5N1-142T infected mice as compared to H5N1-ScrbT and H5N1-126T infected mice (-). Taken together, these results demonstrate that both the direct infection of endothelial cells as well as higher levels of proinflammatory cytokines contribute to increased vascular leakage during H5N1 infection.Prior studies suggest that both proinflammatory cytokines and infection of endothelial cells can increase endothelial permeability, eventually resulting in viral pneumonia ,36,37,38ted mice . As demoted mice  28]. In. In- CD3To understand the pathological changes in the lung upon infection with miR-targeted viruses, we isolated lungs from infected mice and performed hematoxylin and eosin staining (H&E) analysis of lung sections . InteresPrevious studies demonstrate that the multibasic cleavage site (MBS) in HA is essential for the spread of HPAI virus infection to the extrapulmonary organs ,41. To dFerrets are considered an excellent model to study influenza virus pathogenesis, and respiratory epithelial cells are the primary sites of influenza virus replication in ferrets ,43. To ePrevious studies show that H5N1 viruses can spread to the lower respiratory tract of infected ferrets ,44. Our Prior studies have demonstrated that the MBS in HA is critical for the extrapulmonary spread of H5N1 viruses. The removal of the MBS in HA (low pathogenic) restricts H5N1 replication to the respiratory tract ,46; howeHPAI H5N1 virus infection causes severe respiratory distress and can be fatal even in healthy adults. A combination of viral and host factors have been shown to contribute to the rapid progression of disease during H5N1 infection. Here, we investigated if the cell tropism of H5N1 virus is a determinant of pathogenesis by utilizing endogenous miRNA mediated restriction of viral tropism ,28,29. OSeveral prior studies have utilized genetic reassortments of pathogenic and non-pathogenic strains to investigate the basis for the severe disease associated with H5N1 infections ,48,49. HAvian influenza virus strains of the H5 and H7 subtypes can gain an MBS in the HA gene while circulating in terrestrial poultry. An MBS is one of the main determinants of pathogenesis for H5 and H7 strains in several mammalian and avian models, as the MBS allows for the processing of HA by ubiquitously expressed furin proteases and thus permits systemic viral replication ,41,46. ARobust HPAI H5N1 infection has been observed in the reticulo-endothelial system of avian models, and this endothelial tropism has been implicated in high virulence in avian hosts ,25,53. UPrevious studies by us and others show that hematopoietic cells are susceptible to infection by influenza viruses in vivo ,57,58. IIn conclusion, our study demonstrates that endothelial cell tropism is a determinant of the high virulence associated with HPAI H5N1 infection in mammalian hosts. By utilizing endogenous miRNA mediated restriction of viral tropism, we demonstrate that H5N1 infection of endothelial cells results in increased cytokine production in the lungs and loss of endothelial barrier function, which culminates in vascular leakage in the lungs. In addition, extrapulmonary spread of H5N1 virus likely occurs via the hematogenous route by infection of endothelial cells. Importantly, our studies strongly suggest that a combination therapy with drugs that improve endothelial barrier function and suppress host immune responses will be necessary for treating HPAI H5N1 virus infections. Apart from HPAI H5N1 virus, several other influenza virus strains either with (H7N7) or without  an MBS in HA have been shown to cause aggressive disease in mammalian hosts. Future studies are necessary to determine if the high virulence of other pathogenic influenza virus strains is due to endothelial cell tropism.Studies with highly pathogenic H5N1 viruses were performed in a CDC/USDA accredited ABSL3 laboratory by skilled researchers with more than 4 years of experience in the appropriate animal models.All studies were performed in accordance with the principles described by the Animal Welfare Act and the National Institutes of Health guidelines for the care and use of laboratory animals in biomedical research. The protocols for performing mouse and ferret studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Chicago and at the Icahn School of Medicine .Eight week old C57BL/6J female mice were purchased from Jackson Laboratory and housed in an Animal Biosafetly Level 3 (ABSL-3) facility. Mice were anesthetized with ketamine/xylazine and intranasally instilled with the indicated dose of virus diluted in 25μl PBS as previously described . InfecteFive-month old male finch ferrets that tested seronegative for circulating influenza viruses were purchased from Triple F Farms and housed in a ABSL3 facility. Ferrets were anesthetized with ketamine HCl (30mg/Kg) and xylazine (2mg/Kg), and then intranasally infected with 3500 PFU of H5N1 virus diluted in PBS. Ferrets were monitored daily for body weight loss. The humane endpoints for euthanasia included >15% body weight loss or neurological complications.TM -2 media according to the supplier’s instructions (Lonza). Madin-Darby Canine Kidney  cells were maintained in Minimum Essential Medium  supplemented with 10% FBS and Pen/Strep (100 units/mL). THP-1 cells (ATCC) were maintained in RPMI media (Gibco) supplemented with 10% FBS and Pen/Strep (100 units/mL). Bone marrow derived dendritic cells (BMDC) were prepared from the tibia and femur of C57BL/6J mice.Human lung epithelial adenocarcinoma cells , human embryonic kidney cells , mouse lung epithelial cells  and mouse endothelial cells  were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum  and penicillin/streptomycin . Human lung microvascular endothelial cells  were maintained in EGMRecombinant HPAI H5N1 viruses based on a human isolate (A/Vietnam/1203/2004 strain) were generated using the reverse genetics system ,64,65. THEK-293T cells were co-transfected with a vector expressing a specific miRNA and a pDZ vector expressing an NP with miRNA target sites at a ratio of 8:1 using Lipofectamine 2000 reagent (Invitrogen). At 48h post-transfection, the cells were lysed with 1% NP40 lysis buffer and western blot analysis was performed using an NP specific monoclonal antibody . The levels of Ku80 antigen were included as loading controls (Sigma).5/well), HMVEC (1x105/well), THP-1 (4x105/well), LA-4 (4x105/well), J774 (4x105/well) and MS1 (4x105/well) cells were seeded in 12-well plates a day prior to infection. Cells were infected at the indicated MOI calculated based on cell numbers determined prior to infection. Infections were carried out in DMEM/0.2% BSA media for the indicated times and viral titers in the supernatants were determined by plaque assay on MDCK cells. BMDCs (1x106/well) were seeded in 12-well plates, infected at an MOI = 1, and at 24hpi, the levels of infection were determined by flow cytometry using an anti-H5 monoclonal antibody (BEI Resources). Low pathogenic virus replication assays were performed in the presence of 1μg/ml of TPCK-treated trypsin (Sigma).A549  and clarified of debris by centrifugation. Serially diluted supernatants were added to confluent monolayers of MDCK cells seeded in 12-well plates and overlayed with MEM media , 0.1% NaHCO3 (Sigma), 0.01% DEAE-dextran ). After a 48h incubation at 37°C, the cells were fixed with 4% formaldehyde for 1h and plaques were visualized by staining with 0.1% crystal violet solution. Plaque assays for low pathogenic viruses were performed in the presence of 1μg/ml of TPCK-treated trypsin (Sigma).-, CD31+ and viral HA+).Single cell suspensions of mouse lungs were prepared as described previously . BrieflyTotal RNA from the lungs of naïve or infected mice were prepared by homogenizing the entire lung in 1mL Trizol (Life technologies) and followed by extraction using the manufacturer’s recommendations. cDNA was generated by SuperScript II using oligo dT primers (Invitrogen). Real-time PCR was performed on an ABI7300 real time PCR system using SYBR Green (ABI Biosystems). Delta delta cycle thresholds were calculated using tubulin as the endogenous housekeeping gene and data is presented as fold change in expression over naïve mice. Primers used for qPCR analysis have been previously described .C57BL/6J mice were infected with 25PFU of H5N1 virus. On day 7 pi mice were anesthetized with ketamine/xylazine and injected with Evans Blue dye  via retro-orbital route. After 1h, mice were euthanized and bronchioalveolar fluid (BALF) was collected using 1mL of PBS. The levels of Evans Blue dye were determined using a previously established protocol . BrieflyMouse lungs were injected with 1mL of 4% formaldehyde through the trachea and tied-off with suture thread, and kept in formaldehyde for 2 days. Subsequently the lungs were embedded in paraffin, sectioned into 5μM slices and stained with hematoxylin and eosin. Slides were blinded and analyzed by a veterinary pathologist under a microscope.Data were analyzed using Prism GraphPad software and statistical significance was determined by one-way ANOVA. Asterisks *, **, and *** denote a significance with p-Values <0.05, 0.01, and 0.001 respectively.S1 Table(PDF)Click here for additional data file.S1 FigHuman and mouse cell lines were infected at the indicated MOI and at various times post-infection the supernatants were collected and titers were determined by plaque assay on MDCK cells. The titers are shown as PFU/mL (mean ± SEM). The limit of detection is 10 PFU/mL. The cell lines were infected at MOIs: A549 (0.001), THP-1 (0.01), HMVEC (0.01), LA-4 (1), J774 (0.01), and MS1 (1).(TIF)Click here for additional data file.S2 FigC57BL/6J mice (n = 5) were intranasally infected at a dose of 25 PFU and monitored daily for weight loss and survival. Left—Body weight loss, shown as relative percentage of day 0 weight (mean ± SEM) and Right–Survival.(TIF)Click here for additional data file.S3 FigFerret cells were infected at an MOI = 0.001 and at various times post-infection supernatants were collected, and titers were determined by plaque assay on MDCK cells. Left–H5N1 viruses with an HA containing the multibasic cleavage site (High Path). Right–H5N1 viruses with an HA lacking the multibasic cleavage site (Low Path).(TIF)Click here for additional data file.S4 FigHuman and mouse cell lines were infected at the indicated MOI and at various times post-infection supernatants were collected for viral titer determination. The titers are shown as PFU/mL (mean ± SEM). The limit of detection is 10 PFU/mL. The cell lines were infected at MOIs: A549 (0.001), THP-1 (0.01), HMVEC (0.01), LA-4 (1), J774 (0.01), and MS1 (1).(TIF)Click here for additional data file.


Taken together, our results demonstrate the anti-tumor properties of LSD1 inhibition on human AML cell line and mouse xenograft model. Our findings provide mechanistic insights into the LSD1 functions in controlling both differentiation and proliferation in AML.LSD1 (Lysine Specific Demethylase1)/KDM1A (Lysine Demethylase 1A), a flavin adenine dinucleotide (FAD)-dependent histone H3K4/K9 demethylase, sustains oncogenic potential of leukemia stem cells in primary human leukemia cells. However, the pro-differentiation and anti-proliferation effects of LSD1 inhibition in acute myeloid leukemia (AML) are not yet fully understood. Here, we report that small hairpin RNA (shRNA) mediated LSD1 inhibition causes a remarkable transcriptional activation of myeloid lineage marker genes (CD11b/ITGAM and CD86), reduction of cell proliferation and decrease of clonogenic ability of human AML cells. Cell surface expression of CD11b and CD86 is significantly and dynamically increased in human AML cells upon sustained LSD1 inhibition. Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) analyses of histone marks revealed that there is a specific increase of H3K4me2 modification and an accompanied increase of H3K4me3 modification at the respective CD11b and CD86 promoter region, whereas the global H3K4me2 level remains constant. Consistently, inhibition of LSD1  Leukemia is a highly complex disorder of blood and bone marrow, and causes more deaths than any other cancer among children and young adults under the age of 20 . It is c2per day on days 1 to 7 and daunorubicin at 60 mg/m2 per day on days 1 to 3 [Mixed lineage leukemia (MLL) is a genetically distinct form of acute leukemia that constitutes over 70% of infant acute leukemia and approximately 10% of adult AML . MLL diss 1 to 3 , 13. ThuOf 80 different direct and 120 reciprocal MLL fusions, MLL-AF9 is capable of interacting directly, or indirectly, with one histone demethylase, LSD1  , 15. As http://www.proteinatlas.org) [in vitro and in vivo. Serials of validation studies revealed that genetic inhibition of LSD1 results in increased expressions of two myeloid differentiation markers (CD11b and CD86), reduced clonogenicity and decreased proliferation ability in THP-1 and Molm13 cells, while it has no any obvious effect in Jurkat (MLL-WT) cells. Histone 3 lysine methylation profiling by ChIP-qPCR further demonstrated that genetic inhibition of LSD1 increases H3K4me2 and H3K4me3 level on the promoters of CD11b and CD86. The same observation appears on the promoter of CD11b in Molm13 with pharmacological inhibition of LSD1. Additionally, we did not find a clear variation of H3K9me2 modification on those promoters in the same context, which indicates this increased expression of CD11b and CD86, is mainly due to H3K4 modification. Together, our findings underline the possible therapeutic potential of genetic inhibition of LSD1 in MLL-AF9 AML.LSD1 is known to interact with the corepressor complex CoREST, containing RE1-silencing transcription factor (REST) and the histone deacetylases (HDAC) 1 and 2, which augments the gene repression activity of LSD1 . Recent las.org) . Overexplas.org) . High LSlas.org) , 30. Furlas.org) . In addilas.org) . Preclinlas.org) , 32. CD1las.org) . B7-2 (Clas.org) , 35. ColPrevious reports demonstrated that shRNA mediated LSD1 knockdown worked well in a variety of cells –40. To vhttp://cgap.nci.nih.gov/Microarray/GeneList) in several cancer cell lines collected by the National Cancer Institute revealed two myeloid differentiation markers, CD11b and CD86, are potential targeted genes of LSD1 in AML [LSD1 inhibits leukemia stem cell self-renewal and induces their myeloid differentiation [1 in AML , 45, 46.1 in AML –50.LSD1 is a known H3K4 demethylase , 51. To To understand whether LSD1 controls the global H3K4 methylation the LSD1, THP-1-sh1896 and Molm13-sh1896 were treated with Doxycycline (0.5 ug/ml) for 72 hours to induce LSD1 knockdown. Cell lysates were analyzed by western blotting to monitor changes in global H3K4 methylation. We found that global H3K4me2 methylation level remains unchanged after Doxycycline treatment in THP-1-sh1896 and Molm13-sh1896 cells (inducible LSD1 knockdown cell line) Figure . Concordin vitro effects of LSD1 knockdown could be translated into in vivo settings, we established a THP-1-sh1896 xenograft model as described in the material and methods. Treatment with Doxycycline significantly inhibited tumor growth with a T/C ratio of 67%  with an overall incidence of ∼30% . Of overIn this study, we investigated the role of LSD1 in regulating proliferation and differentiation in human MLL-AF9 translocated monocytic leukemia cells. THP-1 and Molm13 cell lines were chosen because THP-1 is the only one AML cell line from a characteristic infant monocytic AML M5 leukemia patient, and Molm13 is the classical cell line established from the peripheral blood of a nine years old boy patient with relapsed acute monocytic AML M5a leukemia. Together, both cell lines have been greatly confirmed to be the most common cellular models for studying monocytic differentiation and leukemogenesis , 70.https://clinicaltrials.gov/; ORY-1001 in phase I/IIa study; GSK2879552 in phase I study). In addition, we noticed that Lynch's group reported that physical association of LSD1 with transcription factors such as GFI1 is essential to maintain the differentiation block in AML and tranylcypromine-derivative inhibitors target this novel scaffolding function of LSD1, rather than its histone demethylase activity, to promote differentiation of AML cells [Previous studies demonstrated that genetic knockdown of MLL-AF9 induced the expression of pro-apoptotic regulators  while reducing the expression of anti-apoptotic regulators , leading to impaired proliferation and apoptosis in THP-1 and Molm13 , 71. In ML cells . It suggML cells , 76–79. ML cells , 81.Taken together, we propose that the anti-AML activity of LSD1 inhibition could be attributed to its function in promoting myeloid differentiation and inhibition of oncogenic proliferation through its demethylation activity Figure . Our finSDS, Glycerol, Tris-HCl, Iodonitro-tetrezolium chloride, Ethanol, Doxycycline (Dox) was purchased from Sigma . All AML cell lines, THP-1, Molm13, Jurkat, HEK293T purchased from ATCC were maintained in RPMI1640 or DMEM medium supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin  and grown at 37°C in 5% CO2 atmosphere.™ HiPerform™ . After overnight, medium was replaced with fresh culture medium. After an additional 72 hours of culture, media were collected and filtered through a 0.45 um syringe filter to remove floating cells, and the aliquot supernatant was stocked in –80°C freezer and used for infection in future.Human LSD1 shRNA-1896 (NM-015013: CCGGCCACGAGTCAAACCTTTATTTCTCGAGAAATAAAGGTTTGACTCGTGGTTTTTG) and shRNA-1970 (NM-015013: CCGGGCCTAGACATTAAACTGAATACTCGAGTATTCAGTTTAATGTCTAGGCTTTTTG) were synthesized and cloned into the pLKO.1 vector with the Puromycin resistant gene according to the manufacturer’s instructions . The 293FT cells were transfected with pLKO.1-LSD1 shRNA-1896 using the Vira PowerCells (8*10E5/mL) were incubated in 6-well plates (Corning) with 1000 uL RPMI-1640 or DMEM supplemented with 10% FBS and 1000 uL of lentiviral supernatant. Polybrene (8 ng/mL) was added to the viral supernatant at a ratio of 1:1,000 (vol/vol). And centrifuged the 6-well plate with 300 g at room temperature (25°C) for 90 min, then cultured at 37°C in 5% CO2 atmosphere for overnight. On the following day, cells were harvested and cultured in RPMI-1640 supplemented with 10 % FBS and Puromycin (1–2 ug/ml) at least for 3 days.All cells are seeded at 1*10E5/5*10E4 with different dose of compounds in 12-well or 24 well plate on day 0 were incubated in RPMI1640 or DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and grown at 37°C in 5% CO2 atmosphere. Refreshed the medium and counted the cell numbers as the protocol by Vi-cell cell viability analyzer system  on different days. All data were finally analyzed by Prism5-GraphPAD.AML cells seeded at 300 per well in a 48-well plate were incubated in human methylcellulose base media with compounds of the required dose for 10–21 days according to the manufacture’s recommendation . Cells were fixed and stained with Iodonitrotetrezolium chloride (8 mg/ml dissolved in ethanol) for above 2 hours, quantitated by Quantity One software in Gel-DocTM XR+ with Image LabTM software system .Cells were treated as required and washed twice with ice-cold PBS, lysed in SDS lysis buffer , applied to western blot analysis. 1–2 ug of total proteins were loaded on 4–12% NuPAGE Bis–Tris gels (Life technology) and transferred onto NC or PVDF membranes by iBLOT transferring system (Life technology). Primary antibodies against Histone H3 was purchased from Sigma-Aldrich  and H3K4me1 (Cat.no. ab8895) and H3K9me2 (Cat.no. ab1220) was purchased from ABcam  H3K4me2 (Cat.no. 07-059) were purchased from Millipore , LSD1 (CST#2139) was purchased from Cell Signaling Technology . β-Actin (Cat.no. A1978) was purchased from Sigma-Aldrich . Secondary antibodies again Rabbit (Cat.No.926-68073) and Mouse (Cat.No.926-32212) were purchased from Li-COR . The Odyssey Infrared Detection System  was used to quantify relative amounts of proteins.Total RNA was prepared by using RNeasy mini kit on Qubic workstation according to manufacturer’s instruction . A total of 0.5 ug RNA was treated with RNase H and then reverse transcribed by using the Superscript III First strand synthesis super-mix for RT-PCR, following manufacturer’s suggestion . The quantity of cDNA was determined by qPCR analysis using Power SYBER green PCR master mix in ABI 7900HT Fast Real –time PCR System . Primer sequences and conditions for qPCR are available upon request.® 488 mouse anti-human CD14 monoclonal antibody , PE-conjugated mouse anti-human CD11b monoclonal antibody , APC-conjugated mouse anti-human CD86 monoclonal antibody  and Alexa Fluor® 647 mouse anti-human CD68 Antibody  are used for FACS staining at 1:200 dilution. FACS samples were acquired using Guava EasyCyte 8HT flow cytometer (EMD Millipore) and CytoFlex S flow cytometer (Beckman Coulter). Cell viability was also examined by FACS analysis with 7-AAD or Fixable Viability dye (Thermo Fisher Scientific) according to the manufacturer‘s instructions. Results were then analyzed with FlowJo V10 software (TreeStar Inc.).Cells were induced LSD1 knockdown by treatment with Dox at 0.5 ug/ml for four days before analysis. Alexa FluorCells were treated with Dox at 0.5 ug/ml for four days prior to analysis. Chromatin shearing was performed using Qsonica Sonicators Q700  to 100–300 base pairs, according to manufacturer’s instructions. ChIP was performed using the Chromatin IP(ChIP) Assay Kit (Millipore)according to standard protocols and the supplier’s directions with ChIP-grade antibodies : anti-H3K4me1, anti-H3 total anti-H3K4me2, anti-H3K4me3 and anti-H3K9me2.We used normal mouse IgG  and normal rabbit IgG as a negative control.6 of Thp1 sh1896 Human Leukemia cell in PBS with 50% matrigel for the tumor development. Measure the tumor volume and body weight twice every week. Tumor sizes will be measured twice weekly in two dimensions using a caliper, and the volume will be expressed in mm3 using the formula: V = 0.5a × b2 where a and b are the long and short diameters of the tumor, respectively. The tumor sizes are then used for the calculations of both T-C and T/C values. T-C is calculated with T as the median time (in days) required for the treatment group tumors to reach a predetermined size , and C is the median time (in days) for the control group tumors to reach the same size. The T/C value (in percent) is an indication of antitumor effectiveness, which is calculated using the formula: T/C% = Ti–T0/Ci–C0 * 100%, Ti is the average tumor volume of a treatment group on a given day, T0 is the average tumor volume of the corresponding treatment group on the day of treatment start, Vi is the average tumor volume of the vehicle control group on the same day with Ti, and V0 is the average tumor volume of the corresponding vehicle group on the day of treatment start. The 2.0 mg/ml Doxy + 1% or 5% sucrose was administrated through the drinking the water.The experiments were conducted on female BALB/c nude mice  aged 4–6 weeks old, weighing approximately 18–22g. The animals were housed in the specific pathogen free animal facility in accordance with the guide for care and use of laboratory animals and the regulations of the institutional animal care and use committee. Each mouse was be inoculated subcutaneously at the right flank with the 5 × 10t-test. All statistical analyses were performed using Prism 5/GraphPad and SPSS 13.0. A value of p < 0.05 was considered statistically significant .All data are expressed as the mean ± standard deviation (SD). Statistical differences between experimental groups were determined using Student’s


The target terthienyls contain a central extended thienopyrazine analogue containing 2-thienyl units at the reactive α-positions of the central thiophene. The extended fused-ring thienopyrazine analogues studied include acenaphthothienopyrazine, dibenzothienoquinoxaline, and thieno-pyrazinophenanthroline. Comparison of the electrochemical and photophysical properties to simple thienopyrazine-based terthienyls and their polymeric analogues are reported in order to provide structure-function relationships within this series of compounds and materials.The synthesis, characterization, and electropolymerization of a series of extended fused-ring thieno[3,4- ThisP[T1]–P[T5] are collected in Epa) and reduction (Epc) processes follow the same basic trends seen in both the monomers [P[T4] is quite sharp, resulting in a determined HOMO energy level that is deeper than expected. The sharper onset and more defined oxidation peak may be indicative of reduced polydispersity, with fewer segments of higher conjugation lengths in the resulting polymer film.The electrochemical data for the resulting electropolymerized polymers monomers  and tertP[T4], all of the HOMO and LUMO energy levels given in g values of P[T3]–P[T5] given here are lower than that reported for all of the copolymeric materials given in b]thienopyrazine  giving lower band gaps , electropolymerization onto ITO resulted in a quite granular deposit, rather than a smooth polymer film as desired. As such, the absorption spectrum exhibits significant scatter evidenced by the long tailing in Eg value of 0.99 eV, which seems to fit the trends previously observed in the terthienyl percursors. The polymers P[T4] and P[T5] exhibit greater differences in absorption and Eg than might be expected from the optical data of T4 and T5. However, if P[T4] contains segments of lower conjugation lengths as proposed above, this could account for the differences observed here for the absorption properties of the polymer films.The extended fused-ring terthienyls P[T3]–P[T5] are lower than that reported for all of the copolymeric materials given in P[T4] with its previously reported analogue P5 . The hib]pyrazine (T1) and 2,3-diphenyl-5,7-bis(2-thienyl)thienopyrazine (T2) were prepared as previously reported  .Terthienyl T4 was obtained as a dark green solid (90%–95% yield). mp ~290 °C (dec); 1H NMR  δ 9.32 , 8.45 , 7.77 , 7.75 , 7.71 , 7.49 , 7.19 . The 1H NMR spectrum is provided in the 13C NMR not obtained due to low solubility; HRMS m/z 450.0320 [M+] .Terthienyl T5 was obtained as a dark turquoise solid (90%–95% yield). mp ~360 °C (dec); 1H NMR  δ 9.15 , 9.10 , 7.55 , 7.47 , 7.40 , 7.08 . The 1H NMR spectrum is provided in the 13C NMR not obtained due to low solubility; HRMS m/z 453.0316 [M + H]+ .Terthienyl T1–T5 were carried out in a three-electrode cell consisting of a platinum disc working electrode, a platinum wire auxiliary electrode, and Ag/Ag+ reference electrode. Solutions consisted of oligomer dissolved in anhydrous CH3CN or CH2Cl2 containing 0.10 M tetrabutylammonium hexafluorophosphate (TBAPF6). The solutions were deoxygenated by sparging with argon prior to each scan and blanketed with argon during the polymerizations. The platinum disc working electrode was polished with 0.05 mm alumina and washed well with deionized water and dry solvent prior to each film growth. The films were grown by cyclic voltammetry scanning through the Epa region for each oligomer.Electropolymerizations of Epa for each monomer until a suitable film was obtained and then held at a fixed potential corresponding to the neutral form of the polymer under investigation.Electropolymerizations for optical experiments were carried out in the same manner as discussed above except an ITO-coated glass plate was used as the working electrode. Polymer films were grown by continuous repeated potential cycling around the + reference electrode ) thienopyrazine (3), dibenzothienoquin-oxaline (4), and thienopyrazinophenanthroline (5) have revealed that these terthienyl species follow the same trends in properties as the respective monomeric units 3–5. In a similar manner, the electropolymerization of these terthienyls provide low band gap materials (Eg = 0.8–1.0 eV) whose polymer films exhibit optical and electronic properties consistent with the trends of both the TP-based terthienyls T3–T5 and the initial monomeric TP analogues 3–5. The fact that these trends hold from monomer to terthienyl to polymer reinforces the claim that the ambipolar nature of TPs and their extended analogues contributes fully to both the HOMO and LUMO of any resulting TP-based materials. As a result, it is thus the electronic nature of the ambipolar TP unit that provides the greatest contribution to the determination of the frontier orbital energies and band gap of TP-based materials.The optical and electrochemical characterization of TP-based terthienyls