K1L and C7L, in conjunction with the removal of the immunomodulatory viral molecule B19, an antagonist of type I interferon action. These novel virus vectors, referred to as NYVAC-C-KC and NYVAC-C-KC-ΔB19R, have acquired relevant biological characteristics, giving higher levels of antigen expression in infected cells, replication-competency in human keratinocytes and dermal fibroblasts, activation of selective host cell signal transduction pathways, and limited virus spread in tissues. Importantly, these replication-competent viruses have been demonstrated to maintain a highly attenuated phenotype.While as yet there is no vaccine against HIV/AIDS, the results of the phase III Thai trial (RV144) have been encouraging and suggest that further improvements of the prime/boost vaccine combination of a poxvirus and protein are needed. With this aim, in this investigation we have generated derivatives of the candidate vaccinia virus vaccine vector NYVAC with potentially improved functions. This has been achieved by the re-incorporation into the virus genome of two host range genes,  K1L and C7L. NYVAC is replication-incompetent in most human cells. While one of the advantages of replication-deficient viruses is their safety profiles, it has been postulated that efficacy of these viruses is limited by the failure to replicate and the attendant limitation in antigen accumulation during virus infection.Following the eradication of smallpox through the use of an efficient live vaccinia virus (VACV) for vaccination, and thereafter, through the discovery of poxviruses as recombinant vectors expressing a large range of foreign genes K1L and C7L host range genes back into the viral genome. Our findings demonstrate that these new constructs (NYVAC-C-KC and NYVAC-C-KC-ΔB19R) have acquired new biological properties distinct from the parental NYVAC-C, which make them potentially improved vaccine vector candidates for human application.Among the many heterologous antigens that have been expressed in these replication-defective poxvirus vaccine vectors are those derived from HIV All animal work at Arizona State University has been conducted in compliance with national and international guidelines. This study was approved by the Institutional Animal Care and Use Committee at Arizona State University, Tempe, Arizona, under Protocol #10-0970R.All animal work at CNB-CSIS has been conducted in compliance with national and international guidelines and with the Royal Decree (RD 1201/2005). These studies were approved by the Ethical Committee of Animal Experimentation of Centro Nacional de Biotecnologia, Madrid, Spain, under Permit numbers 152/07 and 080030.PBMCs for signal transduction assays were obtained from healthy blood donors recruited by the Blood Transfusion Center of Madrid; donations were approved by the ethics commission of the Center and written informed consent was obtained from the donors; all information was kept confidential by the Blood Center.B19R ORF deleted, was obtained by sequential cloning of five DNA fragments containing dsRed2 and rsGFP genes and B19R recombination flanking sequences into the plasmid pGem-7Zf(-) (Promega). The dsRed2 gene under the control of the synthetic early/late promoter was amplified by PCR from plasmid pG-dsRed2 with oligonucleotides Red2-B (GGATCCTAACTCGAGAAA-3′5′-GAACTA) (Bam HI site underlined) and Red2-N (ATGCATTTATTTATTTAGG-3′5′-ATTAGT) (Nsi I site underlined) (785 bp), digested with Bam HI and Nsi I and inserted into the Bam HI/Nsi I-digested pGem-7Zf(-) to generate pGem-Red wm (3740 bp). The rsGFP gene under the control of the synthetic early/late promoter was amplified by PCR from plasmid pG-dsRed2 with oligonucleotides GFP-X (TCTAGAGAGAAAAATTG-3′5′-CGTTGG) (Xba I site underlined) and GFP-E (GAATTCTCAAGCTATGC-3′5′-CTATA) (Eco RI site underlined) (832 bp), digested with Xba I and Eco RI and inserted into plasmid pGem-Red wm previously digested with Xba I and Eco RI to obtain pGem-Red-GFP wm (4540 bp). NYVAC genome was used as the template to amplify the left flank of the B19R gene (364 bp) with oligonucleotides LFB19R-AatII-F (GACGTCGAGAAAGTTAAGAAGATAC-3′5′-TTTTTT) (Aat II site underlined) and LFB19R-XbaI-R (TCTAGATCTTTATTATACGGCACTAA-3′5′-TTTTTT) (Xba I site underlined). This left flank was digested with Aat II and Xba I and cloned into plasmid pGem-Red-GFP wm previously digested with the same restriction enzymes to generate pGem-RG-LFsB19R wm (4871 bp). The repeated left flank of the B19R gene (364 bp) was amplified by PCR from NYVAC genome with oligonucleotides LFB19R′-EcoRI-F (GAATTCGAGAAAGTTAAGAAGATAC-3′5′-TTTTTT) (Eco RI site underlined) and LFB19R′-ClaI-R (ATCGATTCTTTATTATACGGCACTAA-3′5′-TTTTTT) (Cla I site underlined), digested with Eco RI and Cla I and inserted into the Eco RI/Cla I-digested pGem-RG-LFsB19R wm to generate pGem-RG-LFdB19R wm (5194 bp). The right flank of the B19R gene (381 bp) was amplified by PCR from the NYVAC genome with oligonucleotides RFB19R-ClaI-F (ATCGATATATACAATGCATTTTTATATAC-3′5′-TTTTTT) (Cla I site underlined) and RFB19R-BamHI-R (GGATCCAGTTCTATCATAATCATC-3′5′-TTTTTT) (Bam HI site underlined), digested with Cla I and Bam HI and inserted into the Cla I/Bam HI-digested pGem-RG-LFdB19R wm. The resulting plasmid pGem-RG-B19R wm (5545 bp) was confirmed by DNA sequence analysis and directs the deletion of the B19R gene from the NYVAC-C genome.The plasmid transfer vector pGem-RG-B19R wm, used for the construction of the recombinant virus NYVAC-C-ΔB19R, with the 6 BSC-40 (ATCC) cells were infected with an MOI of 0.01 of NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm using Lipofectamine . 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. In the first two passages viruses from selected plaques expressed both fluorescent proteins, in the next two passages viral progeny from selected plaques expressed only one fluorescent marker and in the last two passages viruses from selected plaques do not express any marker due to the loss of the fluorescent marker. The deletion mutant was detected by PCR amplifying of the B19R locus. The resulting NYVAC-C-ΔB19R positive virus plaques were grown in BSC-40 cells, and further passaged twice in primary CEF cells. A P2 stock was prepared in CEFs (Charles River) and used for the propagation of the virus large cultures in CEF cells, followed by virus purification by sequential centrifugation in two 36% (w/v) sucrose cushions in 10 mM Tris-HCl pH 9, and titrated by plaque assay in BSC-40 cells. The purified grown stock of virus was referred to as P3. The B8R locus of NYVAC-C-ΔB19R was deleted in a similar manner.The deletion mutant NYVAC-C-ΔB19R was constructed by fluorescence selection using dsRed2 and rsGFP genes as the selectable markers. 3×10C7L and K1L genes were amplified from the Copenhagen genome using the following primers: NY1, 5′ GTTTGCATCGTGCTTTAACATCAATGG 3′; NY2, 5′ GTCTTACTCATTGCATCGTACGGTTGGCTTATTCCATAGTAGCTTGTG 3′; NY3, 5′ CTACTATGGAATAAGCCAACCGTACGATGCAATGAGTAAGACAATAGG 3′; and NY4, 5′ GTACCTGGCAATAGGTGATAATATGAC 3′. The “KC” fragment was then inserted into the NYVAC-C genome by in vivo recombination (IVR): 150 ng DNA was used to transfect MRC-5 cells (kind gift of Sanofi Pasteur) according to the manufacturer's protocol , in 35 mm dishes; cells were infected with the parental virus, either NYVAC or NYVAC-C, at a multiplicity of infection (MOI) of 0.05 by adding the virus to the transfection mix. After 30 minutes of incubation, 1 ml of OptiMEM with 1% serum was added to each dish. Cells were scraped into the medium at 36 hours post-infection. Following three rounds of freeze/thaw, the IVR scrape was used to infect Vero cells (kind gift of Sanofi Pasteur) to select for viruses competent for large plaque formation in Vero cells. The same method was used to insert KC into NYVAC-C-ΔB19R.The B19R, viral DNA was extracted by the method of SDS-Proteinase K-Phenol from BSC-40 cells mock-infected or infected at an MOI of 5 with wtNYVAC, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning the B19R flanking regions were used for PCR analysis of B19R locus. The amplification reactions were carried out with Platinum Taq DNA polymerase . For verification of the KC fragment, primers NY1 and NY4 were used on viral DNA obtained by standard phenol extraction from BHK-infected cells (ATCC).To test for deletion of To verify the correct expression of the HIV-1 proteins gp120 and GPN from the viruses, monolayers of either BSC-40 or BHK cells were mock-infected or infected at an MOI of 5 with wtNYVAC, NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R, or NYVAC-C-KC-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cell extracts fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody  or polyclonal anti-gag p24 serum  followed by anti-rabbit-HRP  to evaluate the expression of gp120 and GPN proteins respectively.2 atmosphere. At different times post-infection , cells were removed by scraping (lysates at 5×105 cells/ml), freeze-thawed three times and briefly sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.To determine virus-growth profiles, monolayers of CEF cells grown in 12-well tissue culture plates were infected in duplicate at an MOI of 0.01 with wtNYVAC, NYVAC-C or NYVAC-C-ΔB19R. Following virus adsorption for 60 minutes at 37 C, the inoculum was removed. The infected cells were washed once with DMEM without serum and incubated with fresh DMEM containing 2% FCS at 37 C in a 5% COTo compare replication competence in human cells, NYVAC-C, NYVAC-C-KC, and NYVAC-C-KC-ΔB19R were used to infect HeLa cells (ATCC) at an MOI of 5. At 0, 6, 12, and 24 hours post-infection cells were scraped into the medium, pelleted, and the supernatant discarded. The cell pellets were resuspended in 200 µl medium and titers were obtained in BHK cells. Multi-step growth curves were done at an MOI of 0.01 in the indicated cells lines, keratinocytes (Invitrogen) and dermal fibroblasts (Lonza). IFN treatment was 1000 units/ml added for 24 hours prior to infection.HeLa cells and human keratinocytes were infected at an MOI of 5 and allowed to incubate for 6 hours. Cell lysates were prepared by standard methods and analyzed by Western blots probed with antibodies specific to the phosphorylated forms of PKR , eIF2α , and IRF3 (Epitomic), or to the internal control, GAPDH (Abcam).6 PBMCs were mock-infected or infected at an MOI of 5 with wtNYVAC, NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At 4 hours post-infection, cells were removed by pipetting, centrifuged at room temperature for 5 minutes at 3000 rpm, supernatants discarded and cellular pellet resuspended in lysis buffer  containing phosphatase inhibitors. After an incubation of 15 minutes at 4 C, cells were centrifuged at room temperature for 5 minutes at 13,000 rpm and supernatants transferred to new tubes. Denaturation Buffer was added and solution was mixed and denatured for 5 minutes at 100 C. Protein content in samples was measured with a bicinchoninic protein assay reagent kit  and CBA analysis was performed according to manufacturer's recommendations.For human peripheral blood mononuclear cells (PBMCs), phospho-protein levels present in extracts of cells infected with different recombinant viruses were measured simultaneously by Cytometric Bead Array (CBA) using an LSR II flow cytometer (Becton Dickinson). In CBA technology, different bead populations with distinct fluorescence intensities had been coated with capturing antibodies specific for different analytes. These bead populations could be resolved in the fluorescence channels of the flow cytometer. After the beads had been incubated with 50 µl of sample, different analytes in the sample were captured by their corresponding beads. The protein captured beads were then mixed with phycoerythrin-conjugated detection antibodies to form sandwich complexes. Following incubation, washing and acquisition of fluorescence data, the results were generated in graphical format using the BD CBA software. The concentrations of total and phospho STAT1 were measured simultaneously using the Cell Signaling Master Buffer Kit . For this CBA analysis, 4×10Ex vivo derived plasmacytoid dendritic cells pDc,  and myelRNA extraction, amplification, hybridization, and scanning were performed as described previously Gene array analysis: scanned raw data was screened and inspected for quality. Genes with intensities below background in all samples were removed and then minimum-replaced (a surrogate-replacement policy) using the mean background value of the built-in Illumina probe controls. Normalization of chips was done by quantile-normalization.Gene expression data was analyzed using the R software package. Genes are filtered by detection call and by variance filters to allow a reduction in the number of tests and a corresponding increase in power of the differential gene expression analysis All data is MIAME compliant and the raw data has been deposited in a MIAME compliant database.0 DNA content were analyzed by propidium iodide (PI) staining. Briefly, HeLa cells were mock-infected or infected at an MOI of 5 with NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At 24 hours post-infection, cells were centrifuged at room temperature for 10 minutes at 2000 rpm and supernatants discarded. Pellets were resuspended in detergent lysis buffer and cells stained with PI. After an incubation of 30 minutes at 37 C in the dark, the percentage of cells with hypodiploid DNA content was determined using an LSR II flow cytometer (Becton Dickinson). The results are expressed as fold increase in apoptotic cells with respect to uninfected cells.The different stages of cell cycle and the percentage of cells with subGFor studies in newborn mice, pregnant CD1 mice were purchased from Charles River at 10 days gestation. The animals were housed one animal per cage. Intracranial infections with the indicated viruses, using a total volume of 10 µL, were conducted at 48 to 72 hours post-birth of the pups (at least 10 pups per virus), using a 27-gauge needle, as previously described 7 PFU/mouse of WR(TK−), NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At different times post-infection  mice were sacrificed and mouse tissues were processed for plaque assay titration. Peritoneal cells were harvested by mouse peritoneal cavity lavage with 10 ml of sterile PBS, centrifuged at room temperature for 5 minutes at 1200 rpm and stored at −70 C. Spleens, draining lymph nodes, ovaries, and livers were dissected under sterile conditions and stored at −70 C. Peritoneal cells were resuspended in 200 µl complete DMEM media and tissues from individual mice were homogenized in 200 µl complete DMEM media using an Eppendorf-fitted Dounce homogenizer. The production of infectious virus in different mouse tissues was tested by plaque assay in BSC-40 cells. The virus titer was expressed as Plaque Forming Units (PFU) per gram of protein. Protein content in tissue extracts was measured with a bicinchoninic protein assay reagent kit .BALB/c mice were purchased from Harlan. To analyze the biodistribution of virus recombinants in animals, BALB/c were immunized by intraperitoneal route (IP) with a dose of 2×10Another test was also used for biodistribution by measuring levels of antigen expression in mice infected with recombinants NYVAC and NYVAC-KC expressing the luciferase marker. The recombinant viruses WR-luc and NYVAC-luc have been previously described To originally construct the parental NYVAC virus, VACV-Cop had been deleted of eighteen open reading frames (ORFs), including the TK gene B19R (B18R in WR strain), which encodes a soluble type I IFN binding protein B8R, which encodes a soluble type II IFN binding protein B19R gene with a GFP/dsRed selection cassette through the use of in vivo recombination (IVR). The viral genome then resolves to remove the duplicate coding regions, leaving an empty locus in the place of the targeted gene. The process to insert the cassette and remove it is illustrated in B8R, B19R double deletion was prepared in a similar manner, starting with NYVAC-C-ΔB19R. Characterization of NYVAC-C-ΔB19R is shown in B19R was verified by PCR , on the other hand. Insertion of K1L and C7L into NYVAC-C inhibited activation of PKR to background levels seen in VC2-infected cells. Inhibition of PKR activation in NYVAC-C-KC-infected cells was accompanied by an increase of expression of gag-pol-nef protein and gp120 in infected human cells, compared to cells infected with NYVAC-C  was a strong activator of IRF-3 phosphorylation because the E3L protein is not present to mask the dsRNA B19R from either of these viruses resulted in phosphorylation of IRF-3. Results in human keratinocytes . Since deletion of B19R can lead to IFN-induced signaling, we wanted to determine if these viruses affected phosphorylation of IRF-3. IRF-3 is a key transcription factor, which when phosphorylated and activated can lead to induction of type I IFN, and IFN is an important mediator between the innate and adaptive immune systems. inocytes  were simB8R and B19R. The results from infection with this double-deletion mutant were indistinguishable from those of the single deletion of B19R only (data not shown). Infection with either NYVAC-C or NYVAC-C-KC induced very little change to any of the shown IFN-inducible genes, consistent with the IRF-3 phosphorylation and STAT-phosphorylation results shown in B8R and B19R from NYVAC-C results in a strong inflammatory response, as evidenced by the transcriptional up-regulation of all the genes shown on the graph, again consistent with the results seen in K1L and C7L, this response was tempered; infection with NYVAC-C-KC-ΔB8RΔB19R induced increased transcriptional levels of seven of the genes shown, relative to NYVAC-C-KC . The lower level of induction of these genes by NYVAC-C-KC-ΔB8RΔB19R compared to that of NYVAC-C-ΔB8RΔB19R is consistent with signal transduction data, since NYVAC-C-KC-ΔB8RΔB19R induced less IRF-3 and STAT-1 phosphorylation than did NYVAC-C-ΔB8RΔB19R (data not shown).The signal transduction results were extended by microarray analysis of infected cells, as shown in K1L and C7L genes  B19R from NYVAC-C-KC further attenuated the virus by approximately one log, approaching the attenuation of MVA and NYVAC-C. Thus, the safety profile of NYVAC-C-KC-ΔB19R, despite this construct being nearly fully replication-competent in primary human skin cells in culture, is in the same general range as the highly attenuated, replication-restricted vaccinia virus strains, MVA and NYVAC-C.Though our goal was to improve the parental NYVAC vaccine vector, it was equally important to maintain sufficient safety levels with the pox vector. To compare the pathogenicity of NYVAC-C-KC constructs to existing vaccines, we utilized a newborn mouse model, the most sensitive mouse model available for detecting pox virus pathogenesis Since most of the complications due to use of vaccinia virus require spread from the site of inoculation, we followed two approaches to characterize spread of the virus in infected animals: one determining virus titers in different organs and the second, evaluating viral expression by the levels of the marker luciferase in NYVAC recombinants.7 pfu/mouse of virus and at different days post infection the animals were sacrificed and the presence of virus in the peritoneal wash, ovaries, liver, spleen, and lymph nodes was measured. As shown in To measure titers in different organs, mice were inoculated intraperitoneally (IP) with 2×107 pfu/mouse of WR-luc, NYVAC-luc or NYVAC-KC-luc and at different time points, levels of luciferase were evaluated in mouse tissues. As shown in These results were confirmed with recombinant viruses expressing luciferase. Groups of mice were infected IP with 2×10K1L and C7L into the NYVAC genome. Re-insertion of these two genes restored replication capacity in human cultured cells, including physiologically relevant primary keratinocytes and primary dermal fibroblasts. A concern of replication-competence in human cultured cells has been tempered by the demonstration that the replication-competent viruses described in this manuscript are highly attenuated compared to other replication-competent strains. This has been demonstrated through intracranial inoculation of the vectors in newborn mice. Clearly the vector NYVAC-C-KC is highly attenuated compared to the Copenhagen and NYCBH strains  with the vaccine combination of a canarypox virus (ALVAC) and the monomeric protein gp120, giving 31% protection against HIV infection  strains . The attctivated .K1L-C7L into NYVAC-C include the enhanced expression of foreign antigens, such as the HIV-1 protein gp120 and the fusion protein gag-pol-nef in cultured cells  sensors, such as RIG-I and PKR, and initiating signal transduction pathways that may enhance the adaptive immune response against the antigen. The highly-attenuated phenotype of the NYVAC-C-KC-ΔB19R virus, along with higher antigen expression levels than observed with its parental virus NYVAC-C, and its ability to induce pro-inflammatory signaling and pro-inflammatory gene expression, suggest that this virus provides a potential improved vaccine vector for human application.It is curious that deletion of in vitroPrevious studies on the immunological characteristics of NYVAC-C have been documented in different models: mouse


Gossypium mustelinum ((AD)4) is one of five disomic species in Gossypium. Three 45S ribosomal DNA (rDNA) loci were detected in (AD)4 with 45S rDNA as probe, and three pairs of brighter signals were detected with genomic DNA (gDNA) of Gossypium D genome species as probes. The size and the location of these brighter signals were the same as those detected with 45S rDNA as probe, and were named GISH-NOR. One of them was super-major, which accounted for the fact that about one-half of its chromosome at metaphase was located at chromosome 3, and other two were minor and located at chromosomes 5 and 9, respectively. All GISH-NORs were located in A sub-genome chromosomes, separate from the other four allopolyploid cotton species. GISH-NOR were detected with D genome species as probe, but not A. The greatly abnormal sizes and sites of (AD)4 NORs or GISH-NORs indicate a possible mechanism for 45S rDNA diversification following (AD)4 speciation. Comparisons of GISH intensities and GISH-NOR production with gDNA probes between A and D genomes show that the better relationship of (AD)4 is with A genome. The shortest two chromosomes of A sub-genome of G. mustelinum were shorter than the longest chromosome of D sub-genome chromosomes. Therefore, the longest 13 chromosomes of tetraploid cotton being classified as A sub-genome, while the shorter 13 chromosomes being classified as D sub-genome in traditional cytogenetic and karyotype analyses may not be entirely correct.Gossypium mustelinum genome revealed by GISH and 45S rDNA FISH. J. Integr. Plant Biol. 55(7), 654–662.Wu Q, Liu F, Li S, Song G, Wang C, Zhang X, Wang Y, Stelly D, Wang K (2013) Uniqueness of the  Genomicionships . FISH haossypium . We receossypium . Primarin = 4x = 52) cottons have radiated into three lineages since their formation, collectively comprising five species: Gossypium hirsutum ((AD)1), G. barbadense ((AD)2), G. tomentosum ((AD)3), G. mustelinum ((AD)4), and G. darwinii ((AD)5). The extant A and D genome species are most closely related to their diploid (2n = 2x = 26) progenitors 1 and (AD)2 for their economic importance  from species such as G. raimondii (D5) and G. thurberi (D1) as probes to hybridize to mitotic chromosomes of some allotetraploid or diploid species, such as G. hirsutum ((AD)1), G. herbaceum var. africanum (A1-a) (G. barbadense ((AD)2) 4 is the same as those in other allotetraploids. Gossypium D genome species, including (at the least) D5, D1, and G. davidsonii (D3-d) examined in this study. Like in (AD)1 and (AD)2, the GISH-NOR numbers and sites in (AD)4 were identical to or similar as those generated by 45S rDNA. Also very interestingly, there was one pair of very big GISH-NORs in (AD)4, taking the same number and locus as the super-major NOR, which was proved through both mitotic (Early on we reported the GISH-NOR in m (A1-a) , and G.  ((AD)2) . Because mitotic A, C and  mitotic B. Furthe3-d gDNA as probe, the super-major NOR was natural for the species G. mustelinum because two respective images show the same NOR constructions in the two single chromosomes (arrows in 1 and (AD)2, in which NORs or GISH-NORs were all terminal types (When comparing the diamidino-2-phenylindole (DAPI) images A with hyal types .4 mitotic chromosomes with gDNA of G. arboreum (A2) and 45S rDNA as probes. 45S rDNA (2 genome as probe (1 (G. herbaceum) and its wild species (A1-a) in both meiotic metaphase I 2. This also indicates that A genome is more closely related to allotetraploid cottons than D genome. Or stated in terms of species evolution or allopolyploid speciation, A genome contributed more than D genome.One point that should be emphasized is that the hybridization signals of (AD)4 were a focal point in this study. We noted that all hybridization signals in (AD)4 mitotic chromosomes, generated by either gDNA (from both A genome and D genome) or 45S rDNA (1 and (AD)2. In late two disomic cottons, one NOR or GISH-NOR was located in A sub-genome, and the other two in D sub-genome, which indicates that NORs distribute mainly to D sub-genome. Our recent research (data unpublished) demonstrated that NORs or GISH-NORs in two other wild disomic cottons, G. tomentosum ((AD)3) and G. darwinii ((AD)5), were also distributed like (AD)1 and (AD)2. The sole A sub-genomic distribution of NORs makes (AD)4 differ largely from the rest of all disomic cottons, which is somewhat in line with the ITS data by Sub-genome locations of the NORs or GISH-NORs in (AD)45S rDNA E, 4C–E, G. mustelinum based on dual-FISH were used to identified the location of GISH-NOR 4) 4 was huge in size, possibly over 100 times larger than the other two, while the GISH-NORs were relatively similar in the other four AD species. In that case, we could define the large GISH-NOR as a super-major NOR or super GISH-NOR. Secondly, all NORs or GISH-NORs were detected in A sub-genome chromosomes in (AD)4, while one of them were detected in the A sub-genome chromosome and two of them in the D sub-genome chromosomes in the other four allopolyploid cotton species. The huge size and abnormal sub-genome NORs were only found in (AD)4, and not in any other Gossypium species or other plants, including other eukaryotes.Previously, we have reported 45S rDNA loci of  ((AD)4) . In this species . Though 4 with gDNAs of A genome species  as probes were much stronger and higher than those of the D genome species , which was illustrated by GISH images of both mitotic and meiosis chromosomes omosomes –4. Many omosomes , and recomosomes  suggest  studies .4, the 45S rDNA derived from the diploid D donor evolved faster than that of the diploid A donor, and the 45S rDNA of (AD)4 should be similar to the diploid A genome species. Research on sequence data from the ITS and 5.8S rDNA have shown that nearly all rDNA repeats in G. mustelinum, roughly 3,800 in total and each being approximately 10 kb in length, have been homogenized to A-like, with the other allopolyploid species having exclusively D genome-like rDNA ike rDNA . But in ike rDNA B–D, 4A–DG. mustelinum is an alloployploid species; its related diploid donor species G. herbaceum (A1) and G. raimondii (D5) have two or three major 45S rDNA loci  and gene conversion (G. mustelinum ((AD)4) may be different than in other allopolyploid Gossypium species, and unequal crossing over  may play a more important role in the concerted evolution of 45S rDNA in G. mustelinum. This results in all of the 45S rDNA of the D genome donor being “exchanged” to A sub-genome chromosomes of (AD)4, while the 45S rDNA of the A genome donor is transferred to chromosome 3 to form a super-major 45S rDNA loci, and some remaining 45S rDNA becomes the minor 45S rDNA loci in the A sub-genome chromosomes 5 and 9 in (AD)4. Traditional cytogenetic research has indicated that DNA content and chromosome length of diploid A genome species are bigger than that of diploid D genome species, so the 13 longer chromosomes of the tetraploid cotton were named the A sub-genome, while the 13 shorter chromosomes were named the D sub-genome. But to our surprise, in this experiment, chromosomes 12 and 13 of the A sub-genome were actually equal to chromosomes 14 and 16 in length, while chromosomes 1, 2, and 3 of the D sub-genome were longer than chromosome 13 of the A sub-genome, which also holds for G. barbadense . Therefore, classifying the longest 13 chromosomes of tetraploid cotton as the A sub-genome, and the shorter 13 chromosomes as the D sub-genome chromosomes as done in traditional cytogenetic and karyotype analysis may not be entirely correct.In most alloployploid plants, the number of 45S rDNA loci equals the sum of that of their progenitors . HoweverDNA loci , but in nversion . TherefoG. mustelinum is the sole descendent of one branch of the earliest divergence within the AD lineages  endemic to Brazil. Its accession was provided by Dr. Percival of USDA-ARS, Cotton Germplasm Research Unit in College Station, Texas, and was primarily collected from Brazil in 1988 with its collection number being PS 118. It was used only as the target chromosomes in this study.n = 2x = 26): G. herbaceum var. africanum (A1-a), G. arboreum (A2), G. raimondii (D5), G. davidsonii (D3-d), and G. thurberi (D1). The A1-a was provided by the Central Cotton Research Institute of Pakistan in 1986, and the three D genome species were also provided by Dr. Percival. Their accession IP numbers are shown in Table2 is a commercial cultivar, namely Shixiya-1 (SXY-1), and was bred in China in the early 1970s. All these plant materials are being maintained in the Wild Cotton Plantation in Hainan Island, China, or in greenhouses at the Cotton Research Institute, Chinese Academy of Agricultural Sciences, Anyang, China. The 45S rDNA probe was a 8.2 kb Xho-fragment of 45S rDNA, which contained almost a full Arabidopsis rDNA repeat and was prepared from the plasmid JDH 2-15A, which was kindly provided by Drs. Dai and Wu .The probes used in this study include gDNA extracted from following diploid cottons . Images were captured by a charge-coupled device (CCD) system and brought together to make the plate using Adobe Photoshop CS2 software. Images were processed using Adobe Photoshop by changing contrast, brightness and color balance in order to ensure that the whole image was processed uniformly.G. mustelinum as a disomic cotton.For chromosomes adorned with FISH signals, homologous relationships within each cell were inferred on the basis of multiple criteria, including the relative positions of FISH signals on chromosomes, the relative chromosome sizes, chromosome morphology, FISH signal sizes and shapes. The NOR or GISH-NOR positions were interpreted according to the view of


Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H2S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs-926) with NaHS (a H2S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H2S had little effect on eNOS protein expression in ECs. L-cysteine, a precursor of H2S, stimulated NO production whereas blockage of the activity of H2S-generating enzyme, cystathionine gamma-lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3-K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H2S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS-specific siRNA or nitro-L-arginine methyl ester (L-NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H2S on ECs. Our results suggest that H2S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt-dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H2S-induced angiogenesis.Hydrogen sulfide (H Our present study showed that the pro-angiogenic effect of H2S appears to be regulated by both a NO-dependent and an independent mechanism, whereas NO effect on angiogenesis is partially dependent on H2S. We demonstrated that H2S stimulated NO release by increasing eNOS phosphorylation via a p38 MAPK and Akt-dependent mechanism, which contributes to the stimulatory effect of H2S on EC proliferation and angiogenesis.The angiogenic crosstalk between HHuman umbilical vein endothelial cells-derived EA.hy 926 cells were kindly provided by Dr. Cora-Jean S. Edgell Total nitrate/nitrite concentrations were measured by conversion of nitrate to nitrite after incubating supernatants with nitrate reductase (10 U/ml) and NADPH (5 mM) for 1 hr at 37°C. The total nitrite was measured with a Griess assay kit  using a reference sodium nitrate standard curve Endothelial cells were seeded in 6-well plates and cultured until they reached 70–80% confluence. The cells were then transfected with specific siRNA to knockdown CSE or eNOS gene (50 nM). Negative siRNA was used as transfection control (50 nM), using Lipofectamine™ RNAi-MAX transfection reagent according to the manufacturer's instruction (Invitrogen). Overexpression experiments were carried out with plasmid DNA containing CSE cDNA  or eNOS cDNA . Mock empty vector was used as transfection control  g for 15 min. at 4°C. Supernatants were collected, and the same amounts of proteins were separated on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes . All primary antibody incubations were performed at 4°C overnight. The antibody dilution for phospho-eNOS (Ser1177), eNOS, phospho-ERK, ERK, phospho-p38 MAPK, p38 MAPK, phospho-Akt (S473) and Akt was at 1:1000 . Anti-CSE antibody was used at 1:5000 , and anti-β-actin antibody was at 1:10000 (Sigma-Aldrich). The membranes were stripped using a buffer containing 100 mM β-mercaptoethanol, 2% SDS and 62.5 mM Tris-HCl (pH 6.8) at 50°C for 30 min. Membranes were visualized using enhanced chemiluminescence Western blotting system . Densitometric quantification was performed using Alpha Digi Doctor Software . The protein bands were quantified and normalized against either β-actin or total form levels of the target protein, and expressed as a percentage relative to the controls . The phosphorylation level is defined as the ratio between the phosphorylated target proteins and their total forms and expressed in the summarized bar graphs as the percentage of the untreated controls.Cultured cells were collected and incubated in a lysis buffer containing 0.5 M EDTA, 1 M Tris-Cl (pH 7.4), 0.3 M sucrose and a protease inhibitors mixture (Sigma-Aldrich). The cell extracts were sonicated three times (5–10 sec./each) on ice using a cell sonicator  4 cells) were incubated with different agents in 500 μl DMEM and then seeded onto the surface of Matrigel (BD Biosciences). After 12 hrs, the formation of capillary-like structure was imaged by light microscope. The total lengths of tube-like structures per field were measured using image analysis software (NIH Image software- Image J).The Matrigel matrix gel was thawed overnight at 4°C on ice and then added to pre-chilled culture dishes and allowed to polymerize at 37°C for 1 hr. Endothelial cells (2 × 104 cells/well). After 24 hrs of initial seeding, cells were incubated with DMEM serum-free medium for overnight Cells were counted using automated cell counter TC10™ from BioRad  and seeded into 96-well plates (1 × 10Cystathionine gamma-lyase knockout (KO) mice were generated as described previously t-test. Values of P < 0.05 were considered statistically significant.All data were expressed as mean ± SEM. Each data point represented at least three to four independent experiments. Statistical comparisons were evaluated using Student's 2S precursor) on NO production. L-cysteine pre-treatment stimulated NO production in ECs (Stimulation of ECs with NaHS for 30 min. increased NO production over a concentration range from 10 to 100 μM A. The efn in ECs B. Howeven in ECs B. Cystatn in ECs C and D. n in ECs E and F.NaHS (50 and 100 μM) treatment markedly increased the phosphorylation of eNOS in ECs A. The st2S-induced eNOS phosphorylation and the NO production, we examined the roles of Akt, ERK and p38 MAPK in H2S-stimulated NO production. Treatment with NaHS at 100 μM enhanced the phosphorylation of p38 MAPK, Akt and ERK to different levels  treatment stimulated NO production in bovine arterial ECs. While these authors illustrated the H2S-dependent Akt mechanism that stimulates NO production, the involvement of other kinases, like p38 MAPK, or the synergistic partnership between H2S and NO in angiogenesis were not addressed. Conversely, it has been reported that a high concentration of NaHS (300–3000 μM) significantly inhibited the activity of recombinant bovine eNOS Phosphorylation activates eNOS 2S and NO in angiogenesis regulation. Ex vivo aortic explants isolated from CSE-KO mice showed a remarkable decrease in vascular neogenesis when compared to WT mice. L-arginine treatment stimulated angiogenesis in the WT mice and to a lesser extent, in the CSE-KO mice. On the other hand, L-NAME treatment reduced new vessel formation in WT mice, and this inhibitory effect was not significant in CSE-KO mice, suggesting that the angiogenic effect of NO might be mediated through H2S biosynthesis. Cystathionine gamma-lyase overexpression stimulated EC proliferation, whereas CSE knockdown reversed this effect. Interestingly, we found that the pro-angiogenic effect of H2S was partially attenuated in the presence of eNOS inhibitor L-NAME, or after eNOS knockdown using siRNA. Taken together, our results suggest that both gasotransmitters are required for optimal angiogenic activity, yet angiogenesis still proceeds in the presence of either H2S or NO alone albeit to a reduced degree. Previous studies had reported that H2S and NO can mediate angiogenesis without much knowledge about the H2S–NO interaction on angiogenesis 2S and NO is important for physiological control of different vascular function versus rat) and experimental conditions, and we found that H2S and NO, alone or combined, can cause angiogenesis. Hydrogen sulfide-stimulated angiogenesis was partially but not completely inhibited by NO blockage, whereas in CSE-KO mice, NO treatment stimulated angiogenesis but to a reduced level. The exact molecular mechanism underlying H2S-mediated NO pro-angiogenic response is not clear.We explored the possible interaction between H2S promotes NO production in ECs via the activation of a cascade of phosphorylation events, starting from p38 MAPK, Akt to eNOS. Hydrogen sulfide promotes EC tube formation, proliferation and angiogenesis by NO-dependent and independent mechanisms as outlined in 2S may be a key regulator for angiogenic signalling pathways, whether they required NO or not. The elucidation of the H2S–NO relationship in the vascular biology would improve our understanding of the pathogenic mechanisms for cardiovascular disease in general and angiogenic-related diseases in particular.In summary, our studies demonstrate that H


Chronic treatment with a slow-releasing H2S donor, GYY4137, extended median survival by 17-23% and increased tolerance towards oxidative and endoplasmic reticulum (ER) stress. Also, cysl-2, a sulfhydrylase/cysteine synthase in C. elegans, was transcriptionally upregulated by GYY4137 treatment and the deletion of cysl-2 resulted in a significant reduction in lifespan which was partially recovered by the supplementation of GYY4137. Likewise, a mammalian cell culture system, GYY4137 was able to protect bovine aortic endothelial cells (BAECs) from oxidative stress and (H2O2)-induced cell death. Taken together, this provides further support that H2S exerts a protective function which is consistent with the longevity dividend theory. Overall, this study underlines the therapeutic potential of a slow-releasing H2S donor as regulators of the aging and cellular stress pathways. Exogenous hydrogen sulfide (H In animal cells H2S is produced endogenously as a by-product of the transsulfuration (L-cysteine biosynthesis) pathway by the action of two enzymes: cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) . Thoduction -43 and t KO mice , and RNAstinuria ,46 and lstinuria . Collect2S in the pathogenesis of hypertension and atherosclerosis has been provided Click here for additional data file.Table S1www.wormbase.orgFunctional annotation of transcripts measured in Figure 2 (information adapted from ).(DOCX)Click here for additional data file.Table S2GYY4137 mechanism of action investigation using mutant strains (lifespan summary).(DOCX)Click here for additional data file.


Herein we investigated the influence of CTSL activity on the homeostasis of peripheral B-cell populations and bone marrow (BM) B-cell maturation. B-cell numbers were increased in lymph nodes (LN), spleen and blood from CTSLnkt/nkt mice. Increases in splenic B-cell numbers were restricted to transitional T1 and T2 cells and to the marginal zone (MZ) cell subpopulation. No alterations in the proliferative or apoptosis levels were detected in peripheral B-cell populations from CTSLnkt/nkt mice. In the BM, the percentage and the absolute number of pre-pro-B, pro-B, pre-B, immature and mature B cells were not altered. However, in vitro and in vivo experiments showed that BM B-cell production was markedly increased in CTSLnkt/nkt mice. Besides, BM B-cell emigration to the spleen was increased in CTSLnkt/nkt mice. Colony-forming unit pre-B (CFU pre-B) assays in the presence of BM stromal cells (SC) and reciprocal BM chimeras revealed that both BM B-cell precursors and SC would contribute to sustain the increased B-cell hematopoiesis in CTSLnkt/nkt mice. Overall, our data clearly demonstrate that CTSL negatively regulates BM B-cell production and output therefore influencing the homeostasis of peripheral B cells.Cathepsin L (CTSL) is a ubiquitously expressed lysosomal cysteine peptidase with diverse and highly specific functions. The involvement of CTSL in thymic CD4+ T-cell positive selection has been well documented. Using CTSL B-cell development occurs continuously during life. In adult mice, this process is initiated in the bone marrow (BM) where hematopoietic stem cells differentiate through a series of intermediate stages during which cells are thought to become progressively more restricted in their developmental potential. Once the B-lineage restricted stage is reached, B-cell progenitors execute a programmed development, first rearranging the immunoglobulin heavy chain gene at the pro-B stage, then undergoing multiple rounds of clonal expansion at the pre-B stage and finally rearranging the light chain gene to yield newly formed B cells expressing surface IgM. These immature B cells are exported primarily to the spleen where they progress through stages of immature transitional B cells and develop into mature naïve B cells Cathepsin L (CTSL) is an abundant and ubiquitously expressed lysosomal cysteine peptidase which degrades a wide range of cytoplasmic and nuclear proteins nkt/nkt mice -which carry an inactivating mutation in the Ctsl gene Several cathepsins contributed in the processing of both antigens and self-antigens to antigenic peptides nkt/nkt mice are enlarged and show an increased number of lymphocytes. In spite of the low rate of CD4+ cell thymic production, the number of LN CD4+ T cells is similar to that of wild-type (wt) mice due to a marked increase in their proliferative level. In addition, the number of LN CD8+ cells is significantly increased correlating with an increased thymic export of CD8+ cells Lymph nodes (LN) from CTSLRecently, a role for cathepsin B in B cell development has been proposed nkt (CTSLnkt/nkt), BALB/c and CByJ.B6-Tg (UBC-GFP) 30Scha/J transgenic BALB/c expressing enhanced green fluorescent protein (GFP) under the direction of the human ubiquitin C promoter (BALB/c.GFP). The origin and development of the CTSLnkt/nkt congenic (N>12) strain has been previously described nkt/nkt mice were identified by their alopecy and by the presence of a deletion in both copies of the Ctsl gene. The deletion was detected by RT-PCR  as previously described The following specific pathogen-free mice were used: BALB/c.Cg-CtslHousing and breading in our animal facility  and all experimental procedures were carried out according to the policies of the Academia Nacional de Medicina, based on “Guide for Care and Use of Laboratory Animals. Bethesda, MD: National Institutes of Health; 1985. NIH publication N.85-23”. Experiments were approved by the ethical committee of the IMEX-CONICET (Permit number 1009).LN cell suspensions were prepared from a pool of axillary and inguinal LN as previously described Anti-murine mAb directly conjugated to FITC, PE, Cy, TruRed or APC were used for fluorescence-activated cell sorting. FITC-labeled mAb included anti-HSA (M1/69), anti-IgM (R6-60.2), anti-IgD (11-26c.2a), and anti-B220 (RA3-6B2). PE-labeled antibodies included anti-CD19 (1D3), anti-CD43 (S7), anti-B220, anti-CD23 (B3B4) and anti-IgM. Cy-labeled anti-B220, TruRed-labeled anti-IgD and APC-labeled anti-CD21 (7G6) mAb were used. All antibodies were purchased from BD Pharmingen  except for TrueRed anti-IgD which was purchased from Biolegend .6) from BM, LN or spleen resuspended in RPMI 1640 without phenol red (GIBCO) containing 3% FBS, 0.1% sodium azide, and 10 mM HEPES (GIBCO), were incubated in one step with the appropriate mAb Cells  for 2 days. Additional mice injected with sterile PBS were used to determine background values. Cells from BrdU-treated mice were stained for surface expression of IgM, IgD, CD43 and B220, washed, fixed and permeabilized using BD Cytofix/Cytoperm Buffer. Then cells were washed, treated with 30 µg of DNase I (Sigma), washed and stained with anti-BrdU-FITC before FACS analysis Propidium iodide (PI) (Sigma) or Annexin V (BD Pharmingen) were independently used to detect apoptotic B cells. For PI staining, cells resuspended in PBS containing 3% FBS were incubated with FITC-labeled mAb directed against surface markers. Cells were washed, fixed with 1 ml of ethanol 70% and stored at 4°C for 24 h. Then, cells were washed and resuspended in PBS containing 1% glucose, 1 mg/ml RNase A (Sigma), and 20 µg/ml PI. After 30 min of incubation, cells were acquired on a FACScan flow cytometer For DNA content analysis, PI or 7-amino-actinomycin-D (7-AAD) (BD Pharmingen) g for 5 min  and stained with May-Grunwald Giemsa.B220+ cells from LN were purified by positive selection by using CD45R (B220) MicroBeads  according to the manufacturer's protocol. The B cell purity  was determined by flow cytometry. Cytocentrifuge slides were prepared by centrifugation of samples at 80 nkt/nkt mice were exposed to 500 rad of ionizing radiation and BM and spleen B-cell subpopulations were analyzed by FACS thirteen days post-irradiation Wt and CTSL5) were cultured in 35-mm2 plastic Petri dishes containing 1 ml of the recombinant IL-7 (rIL-7)-supplemented MethoCult M3630 medium . Colonies containing more than 30 cells were scored under an inverted microscope after culture for 7 days at 37°C in a fully humidified atmosphere with 5% CO2Femoral BM cells , 1% L-glutamine, at 37°C in a humidified atmosphere at 5% COnkt/nkt mice were cultured at 1×106 cells/ml in DMEM supplemented with 20% FBS, 2×10−5M 2-ME, 1% L-glutamine, and 2 ng/ml of murine rIL-7 (R&D Systems) and plated in six-well culture trays. Nonadherent cells were harvested after 4 days of culture. This bulk culture provided a highly rich source of IL-7-responsive pro-B cells (>70% B220loCD43+HSAhi). Pro-B cell-rich populations were suspended at 5×105/ml in DMEM supplemented with 20% FBS, 0.1% ME, 1% L-glutamine and 1 ng/ml of murine rIL-7. Aliquots (1.0 ml) of this cell suspension were added to established SC monolayers in 24-well flat-bottomed trays and co-cultured at 37°C in a fully humidified atmosphere of 5% CO2. Nonadherent cells were harvested after 3 days, counted, and cloned using the CFU-pre-B colony assay system.To obtain a pro-B cell-rich population a bulk culture of pooled BM cells stimulated with rIL-7 was performed as described by Tsuboi el al 6 BM cells from wt, CTSLnkt/nkt were i.v. injected into 8-week old mice that had received 900 rad of whole body irradiation 24 h previously. Parallel experiments using BALB/c.GFP instead of wt mice were developed in order to confirm the B-cell donor-origin in reconstituted mice. 2 days before and following irradiation, mice were given drinking water supplemented with 2 mg/ml neomycin sulfate. Twelve days after adoptive transfer, BM and spleen cells were harvested from recipient mice for analysis.Reciprocal BM chimeras were constructed as described by Mollenn et al. t test was used to assess the statistical significance of the results. Results are expressed as (mean ± SD). A value of p<0.05 was considered indicative of a significant difference. Except where specified, the figures and tables contain representative results from experiments repeated independently 3 times.The two-tailed Student´s nkt/nkt mice showed alterations in the number of B cells in the enlarged LN of these mice. As can be observed in F1g 1A, the percentage of B220+cells was significantly increased in LN of CTSLnkt/nkt mice. Both the percentage and absolute number of B220+CD19+LN cells were markedly increased in CTSLnkt/nkt mice (lo IgDhi whereas IgMhi IgD+/hi cells were not detected (data not shown) indicating that LN B cells of CTSLnkt/nkt mice are phenotypically mature. No significant morphological differences were detected between B cells from wt and mutant mice . Neither B-cell basal apoptosis levels were modified as measured by PI staining , thus indicating that alterations in proliferation or apoptosis levels are not involved in the increase in the number of LN B cells in mutant mice.PI staining revealed no alterations in B-cell basal proliferative levels in LN from CTSLhiB220lo/+). Based on HSA and CD21 expression, the analysis of B splenic subsets revealed that the percentages of T1 (CD21loHSAhi) and T2+MZ (CD21hiHSAhi) B cells were increased in CTSLnkt/nkt mice as compared to wt mice (intHSAlo) B cells was decreased in mutant mice. Analysis of CD23 expression in CD21hiHSAhi cells (T2+MZ) showed that the proportion of T2 and MZ B cells was similar in wt and CTSLnkt/nkt mice, indicating that both subsets were equally increased in mutant mice . In addition, no differences were observed in the apoptosis levels .Incorporation of 7-AAD was used to measure the proliferation nkt/nkt mice (hiB220lo/+) and involved both transitional T1 (IgMhiIgDlow) and T2 (IgMhiIgDhi) B-cell stages . Regarding the mature B-cell population (IgMloIgDhi), no differences in the absolute numbers were detected between normal and mutant mice .Regarding peripheral blood, B-cell levels were also increased in CTSLnkt mice . As in tnkt/nkt mice, the number of transitional B cells is increased.Thus, both in the spleen and peripheral blood of CTSLnkt/nkt mice. Taking into account that BM pro-B and pre-B cells proliferate actively + B cells were detected, the absolute number of BrdU-labeled transitional and mature B cells was increased in the spleen of CTSLnkt/nkt mice, indicating that the entry of newly formed B cells into the spleen is increased in these mice.The fact that splenic transitional B cells were increased in the absence of alterations in their proliferative and apoptosis levels led us to investigate whether BM B-cell emigration to the spleen was increased in CTSLnkt/nkt mice. No differences in total BM cellularity or in total BM leukocyte numbers were detected between normal and mutant mice (loCD43+HSA−/lo), pro-B (B220loCD43+HSAhi), pre-B (B220loCD43−IgM−), immature (B220loCD43−IgM+) and mature B cells (B220hiCD43−IgM+) were found to be similar in wt and mutant mice  nkt/nkt mice. This increase included all BM B-cell developmental stages, except for mature B cells which at this time point were undetectable both in mutant and in wt mice. Regarding the spleen, the percentage and absolute number of transitional B cells (HSAhiB220lo/+) was about 7 fold higher in CTSLnkt/nkt mice as compared to wt mice (hiIgDlow) and T2 (IgMhiIgDhi) B-cell subsets , a small number of colonies was obtained even when high numbers of nonadherent BM cells were used. We thus assayed a method described by Tsuboi et al. nkt/nkt mice were co-cultured with wt or CTSLnkt/nkt BM SC and the differentiation potential of conditioned progenitor cells into pre-B cells was then assayed. After co-culture with wt SC, the number of pre-B-cell colonies derived from CTSLnkt/nkt B-cell precursors was 2 fold higher than that derived from wt B cell precursors  BM and spleen cells were analyzed twelve days after transplantation by flow cytometry. As can be observed in nkt/nkt recipients of BALB/c.GPF BM cells were GFP+, indicating that the reconstituting B cells were derived from GFP+ donor BM cells. As can be observed in nkt/nkt recipients of wt or BALB/c.GFP+ cells showed higher absolute numbers of precursor-B cells than wt recipients. Regarding the spleen, both the percentage and absolute numbers of transitional B-cells were higher in CTSLnkt/nkt recipients . These results indicate that the absence of CTSL activity on BM microenvironment influences BM B-cell production and the number of transitional B splenocytes.In order to evaluate cipients . Two mo nkt mutation on BM hematopoietic progenitor-cells is also able to influence in vivo B cell lymphopoiesis, BM cells from wt or CTSLnkt/nkt mice were transferred into lethally irradiated wt mice. As can be observed in nkt/nkt BM cells had higher percentages and absolute numbers of BM B-cell precursors than those that received wt BM cells. These mice also showed increased percentage and absolute numbers of transitional (T1 and T2) splenic cells  and in vivo (autoreconstitution experiments). Of interest, no alterations in the total number of B-cell precursors were detected. In addition, their proliferative or apoptosis levels were neither altered (data not shown). Based on BrdU pulse-chase experiments, we could demonstrate that higher numbers of immature B cells leave the BM and reach the spleen of CTSL mutant mice.Regarding the BM, a clear increase in CTSLnkt/nkt mice leading to an increase in the number of peripheral B cells, notably in follicular LN B-cells.Collectively, our data show that both BM B-cell production and emigration are increased in CTSLin vitro data led us to hypothesize that the nkt mutation could influence B-cell production by acting both on BM SC and BM B-cell precursors. To confirm this hypothesis, in vivo reciprocal BM transplantation experiments were performed. Our results showed that CTSLnkt/nkt hosts receiving wt or GFP.BALB/c cells had higher numbers of BM B-cell precursor and transitional B-splenocytes as compared to wt hosts, thus confirming that the nkt mutation influences B-cell production by altering the BM SC. Besides, the lack of CTSL activity would also affect the intrinsic ability of BM progenitors to develop into B cells, as indicated by the increase in the number of both BM B-cell precursors and transitional B splenocytes in wt mice transplanted with BM mutant cells.It is known that BM SC regulates BM B-cell development through the secretion of various cytokines, growth factors and hormones that affect progenitor cell growth, differentiation, and/or survival Taken together, these findings point both to alterations in BM SC and in hematopoietic progenitor cells as contributing to the increased B-cell hematopoiesis in mutant mice.nkt/nkt mice remain to be defined. To date, CTSL activity on factors known to be involved in B-cell hematopoiesis has only been studied in tissues and organs other than BM. Of note, it has been reported that CTSL-deficient skin fibroblasts show a decreased degradation of insulin-like growth factor binding protein-3 (IGFBP-3) from insulin-like growth factor-1 (IGF)/IGFBP complexes nkt/nkt mice would be directly involved in the increases registered in BM B-cell production. Moreover, decreased degradation of IGFBP-3 from IGF/IGFBP complexes in CTSL-deficient fibroblasts also results in an extracellular accumulation of IGFBP-3 in vitronkt/nkt mice.The molecular mechanisms that underlie the increased BM B-cell production in CTSLIn a model of myocardial infarction, it has been shown that the expression of stem cell factor and stromal cell-derived factor-1 were significantly decreased in the myocardium of CTSL KO mice nkt/nkt B-cell precursors and/or BM endothelial cells could be involved in the increased BM B-cell hematopoiesis.On the other hand, it has also been recently shown that CTSL is able to block AKT signaling in cardiomyocytes Even though there is accumulating evidence for specific functions of CTSL in health and disease, to our knowledge this is the first report showing that CTSL plays an important role in the regulation of the homeostasis of the B-cell pool. As schematized in


A minus sign was omitted between pre-RT and W. Please view the complete, correction equation here:


Tat protein, released by HIV-infected cells, has a battery of important biological effects leading to distinct AIDS-associated pathologies. Cell surface heparan sulfate protoglycans (HSPGs) have been accepted as endogenous Tat receptors, and the Tat basic domain has been identified as the heparin binding site. However, findings that deletion or substitution of the basic domain inhibits but does not completely eliminate Tat–heparin interactions suggest that the basic domain is not the sole Tat heparin binding site. In the current study, an approach integrating computational modeling, mutagenesis, biophysical and cell-based assays was used to elucidate a novel, high affinity heparin-binding site: a Lys12, Lys41, Arg78 (KKR) spatial domain. This domain was also found to facilitate Tat-driven β1 integrin activation, producing subsequent SLK cell adhesion in an HSPG-dependent manner, but was not involved in Tat internalization. The identification of this new heparin binding site may foster further insight into the nature of Tat-heparin interactions and subsequent biological functions, facilitating the rational design of new therapeutics against Tat-mediated pathological events. The transactivating factor of the HIV-1 virus (Tat), released from human immunodeficiency virus type 1 (HIV-1) infected cells, has been pointed towards a variety of important biological functions related to the distinct AIDS-associated pathologies in AIDS patients, including neuropathiesTat is a polypeptide of 86–102 amino acids depending on the viral strainTat-driven activities depend precisely on its interaction with cell surface heparan sulfate (HS) and heparin through their negatively charged sulfate groupsToward this end, we sought to identify novel Tat heparin binding sites using the molecular simulation for appreciable prediction, combined with the surface plasmon resonance (SPR)-based competitive inhibition assay and cell-based functional evaluation with the introduction of several relevant targeted mutant Tat proteins. Encouragingly, a triad of basic residues—Lys12, Lys41 and Arg78 (KKR)—that are spatially enclosed rather than sequentially oriented, was thus identified as a hereto unrecognized high-affinity heparin binding site which help facilitate Tat-driven β1 integrin activation and subsequent adhesion in an heparan sulfate protoglycan (HSPG)-dependent manner. Clearly, our findings will facilitate a crucial touch on a precise understanding of the comprehensive roles of Tat as well as aid in design of new therapeutic agents.ζ, which itself is engaged in two tight salt bridges . Nζ atom of Lys12, in a like manner, was involved in two hydrogen bonds, established with the 3-O (3.30 Å) and 2-O (2.74 Å) of the glucuronic acid ring. Another strong salt bridge (2.77 Å) was observed between the 5-carboxylate group and Nη of Arg78. The binding free energy of the disaccharide was further calculated as −8.84 kcal/mol, indicative of the nanomolar or even lower level of the binding affinity of Tat-III for heparin.Computational modeling was first employed with the aim of identifying the potential heparin binding sites. Here, the homology model of Tat protein (Tat-III) constructed on the basis of NMR data was utilized as the target structure, and several heparin oligosaccharides including di-, tetra-, hexa-, and octa-saccharides were selected as docking probes. In the disaccharide heparin model, heparin was noted to preferentially interact with a triad of basic residues ; Of particular note, among the 20 distinct docking runs, though a few predicted poses were docked at the basic domain, it however generated much higher estimated binding free energies. These finding from an alternative view suggested that the KKR triad may yield a higher affinity for heparin than that of the basic domain.Modeling of interactions with tetra-, hexa- and octa-saccharides revealed similar results; the positively charged KKR triad plays an important role in the molecular recognition of heparin probes producing a highly conserved interaction with Tat-III. It should be noted that the oligosaccharide chain begins to stretch toward the basic domain (yellow for comparison) as the probe length increases. As the length increases to a tetramer, a delicate interaction with residue Arg49 of the basic domain is established; the hexamer follows the same path and approaches residue Arg52; when the heparin oligosaccharide reaches octameric size, it enlists residue Gln54. Their binding free energies were calculated as −9.21, −11.53 and −12.30 kcal/mol, respectively. Heparin ideally, if long enough, is proposed to encompass both the KKR region and fully extend to the whole basic domain; and Arg49 might serve as a ‘knot/link’ between the two binding sites.E. coli as GST fusion proteins, purified from the bacterial lysates by glutathione-agarose affinity chromatography and checked by SDS-PAGE  were injected over a heparin-immobilized sensor chip surface. RUmax values were generated using BIAcore evaluation 3.1 software, yielding an EC50 value of 1.5 µM A/R78A] displayed a markedly decreased binding potency for heparin when compared to wild-type GST-Tat ((G48-R57)A/KA/R78A led to the complete abolishment of Tat-heparin interactions. These findings substantiate our hypothesis that Tat indeed possesses another heparin binding site, identified herein as the KKR region.We next investigated whether KKR is a heparin binding site, as predicted. A series of comparative studies were performed using all relevant mutants. We found that GST-Tat binding to heparin involves multi-phasic binding [13], ind GST-Tat . MoreoveD values were calculated using BIAcore evaluation 3.1 software. Mutation of a single residue  led to only a partial loss of heparin binding affinity , with the Arg78 mutation (GST-TatR78A) influencing binding affinity the least. Moreover, mutation of any two KKR residues A, GST-TatK12A/R78A, GST-TatK41A/R78A) led to a significant loss of binding affinity with correspondingly decreased KD values , with Lys12, Lys41 mutation A) affecting binding the most. Most significantly, concurrent mutation of all three residues produced a dramatic drop in heparin binding affinity, with a resultant KD value of 32.6 nM. This series of experiments collectively indicated that Lys12, Lys41 and Arg78 cooperate to profoundly impact heparin-binding , a key feature of the binding mode. A series of concentrations of GST-Tat, GST-Tat(G48-R57)A and GST-TatK A/R78A were injected over the heparin-immobilized sensor chip surface (D = 1.17 pM) (D = 5.31 pM). This value represents nearly a five-fold reduction in affinity as compared to wild-type GST-Tat. Mutation of the KKR region, however, caused a remarkable decrease in GST-Tat-heparin binding (KD = 32.6 nM), representing the KD value of the basic domain in isolation. This KD represents a 10,000 fold decrease in binding affinity compared with that of wild-type GST-Tat (We next sought to delineate the nature of KKR-associated heparin binding in an attempt to define the intrinsic properties of this newly discovered binding site. We studied binding affinity (K surface . We foun1.17 pM) . Mutatio GST-Tat . Based o(G48-R57)A) produced an IC50 value (0.659 µg/ml) comparable to that of GST-Tat (0.717 µg/ml), while mutation of the KKR region A/R78A) yielded an IC50 value of 1.574 µg/ml, almost twice that of GST-Tat. These data further support the notion that the KKR spatial domain contributes dramatically to heparin binding, and represents an extremely high-affinity heparin binding site.We next conducted a series of experiments evaluating heparin-mediated competitive inhibition of Tat-heparin binding for further confirmation. Wild-type GST-Tat or the appropriate Tat mutants (1.5 µM) was injected over a heparin-immobilized chip in the presence of varying concentrations of heparin as a competitive inhibitor. Free heparin was able to inhibit the binding of either GST-Tat or the corresponding mutant to immobilized heparin in a dose-dependent manner . Notably50 values of 0.717 µg/ml and 20.677 µg/ml respectively. By contrast, other GAGs including CSA, CSC and HA, even at concentrations up to 250 µg/ml, showed little or no inhibition of this interaction  using competitive inhibition assay. Both heparin and HS were able to compete for KKR binding to heparin, yielding ICeraction . These fThus, the newly identified KKR region, distinct from the well-recognized linearly contiguous basic domain, serves as both an extremely high-affinity and highly specific binding motif for heparin.(G48-R57)A) is expected to cause a complete loss in Tat internalizationKA/R78A), however, failed to affect internalization at all. This helps exclude the involvement of the KKR spatial domain in Tat protein internalization.It is well-known that Tat-regulated function mostly precisely depends on the association of Tat with cell surface heparin sulfate/heparin via the negatively charged sulfate group. Of note, Tat-driven internalization indispensably counts on Tat's heparin-binding propertiesKA/R78A) induced a partial but incomplete abolishment of SLK cell adhesion compared with that of GST-Tat (R78A/G79A/D80A) resulted in a complete defect in cell adhesion. These findings together underscore the predominant role of RGD on one hand and meanwhile revealing a complementary action of KKR in Tat-driven cellular adhesion. Furthermore, mutation of the basic domain (GST-Tat(G48-R57)A) generated a similar profile, as did double mutation of both KKR and the basic domain (GST-Tat(G48-R57)A/KA/R78A) which yields a more potent reduction in cell adhesion compared with KKR-mutated Tat. These findings further support the concept that the KKR spatial triad is an essential, but incomplete, contributor to Tat-mediated cell adhesion.In addition to the Tat-driven internalization, Tat also mimics the effect of extracellular matrix molecules and favors cell adhesion via the amino acid sequence RGD GST-Tat , suggestKA/R78A. GST-Tat(G48-R57)A treatment resulted in conserved diffuse β1 integrin staining, while GST-Tat(G48-R57)A/KA/R78A produced less β1 integrin activation. However, the activated β1 integrin induced by GST-TatR78A/G79A/D80A couldn't be detected due to its disability on cell adhesion (data not shown). All these findings are concordant with the results obtained in the cell adhesion assay, which is the consequence of β1 integrin activation.Since Tat promotes SLK cell adhesion through binding α5β1 integrins(G48-R57)A]. Consistently, MAB2040 also significantly blocked SLK cell adhesion facilitated by KKR-dominant but basic domain-substituted Tat [GST-Tat(G48-R57)A], indicating that KKR-mediated adhesion is heparin-dependent.To address whether cell surface HSPG acts as a chaperone for KKR-facilitated cell adhesion events in the context of its heparin binding properties, we performed a series of competitive inhibition assays designed to test its effects on cell adhesion. This was accomplished either by introducing soluble heparin or a specific monoclonal antibody raised against cell surface HS (MAB2040). GST-Tat-induced SLK cell adhesion was inhibited by heparin at concentrations of 200 µg/ml . Further(G48-R57)A] A] . TogetheRusnati et al.In the present study, a homology model of Tat-III constructed using its NMR structure docked with heparin oligos probes provided a promising candidate motif for heparin binding in addition to the well-recognized basic domain. Further analysis of the free energy of heparin-Tat-III interactions and detailed assessment of their atomic associations, coupled with flexible molecular docking schemes substantially predicted the confluence of a triad of basic residues Lys12, Lys41, Arg78 (designated as KKR triad) into a previously unrecognized binding site for heparin.The SPR biosensor assay is an accurate and powerful method of dig the molecule-molecule interactionsD value) was calculated as 5.31 pM, which represents an extremely high-affinity binding interaction . The disparity in binding affinity between the KKR and the basic domain, in principle, should allow heparin to preferentially occupy KKR. In silico simulations clearly demonstrate that heparin can specifically recognize the KKR region when the disaccharide probe was used. As the length of the oligosaccharide increases, the oligomer stretches toward and along the basic domain: it begins to form interactions with Arg49 when the tetramer probe was used, approaches Arg52 as the probe size increases to 6 sugar unit, and reaches Gln54 as the sugar length is 8. Ideally, heparin at its typical length would simultaneously bind to both KKR and the whole basic domain, with residue Arg49 serving as the ‘knot/link’ between the two regions.Delineation of the intrinsic properties of the newly identified binding site is important since it assists in unraveling the nature of the domain's interaction with heparin. By kinetics analysis, the dissociation constant  and C (CSC), hyaluronic acid (HA) and Cy3-conjugated sheep anti-mouse IgG were obtained from Sigma . Anti-GST antibody (sc-459) was purchased from Santa Cruz Biotechnology, Inc. . Monoclonal antibodies to activated-integrin β1 (12G10) and HS (MAB2040) were obtained from Chemicon International, Inc. . FITC-conjugated goat anti-rabbit IgG was purchased from Jackson Immunoresearch Laboratory Inc. .2.SLK cells  were kindly provided by the NIH AIDS Research division. The cells were maintained in RPMI1640 medium supplemented with 10% heat-inactivated fetal calf serum , 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified incubator with 5% COhttp://www.ncbi.nlm.nih.gov/) (GI: 328765) for phylogenetic analyses. A BLASTP (http://www.ncbi.nlm.nih.gov/blast) search of the sequence revealed a highly homologous protein, Tat-I , of which 11 NMR solution structures have been determined (PDB entry 1JFW)The amino acid sequence of human T-lymphotropic virus 3 (Tat-III protein) was retrieved from the appropriate NCBI Pubmed entry  and pGST-Tat were kindly provided by Profs. M Giacca and M Prestai . Tat mutants were constructed using site-directed mutagenesis, yielding series of GST-Tat mutants .The plasmids expressing different Tat mutants were obtained by cloning a PCR-amplified fragment into the BamHI and EcoRI sites of the vector pGEX-2T. Templates for amplification were plasmid pGEX2T-Tat-derived. All the constructs were verified by DNA sequencing. Recombinant wild type HIV-1 Tat and the different Tat mutants were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and purified to homogeneity from bacterial lysates by glutathione-sepharose affinity chromatography (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The purity and integrity of the protein product were routinely checked by SDS-polyacrylamide gel electrophoresis and 0.25% Coomassie brilliant blue R-250 staining. The purified proteins were stored in aliquots at −80°C until useTo characterize Tat binding with heparin, kinetic properties and competitive inhibition were examined using a surface plasmon resonance assay . For this purpose, heparin was immobilized to CM5 sensor chip according to established method5 cells/ml in RPMI1640 with 10% FCS). After 12 h, GST-Tat or the relevant mutant protein (1.6 µg/ml) was added to the cell culture medium and incubated for 24 h. Cells were then washed three times with phosphate buffered saline (PBS) and fixed with cold 4% (v/v) paraformaldehyde for 30 min at room temperature. Subsequently, the cover slips were washed with PBS, permeabilized for 15 min in PBS containing 0.1% (v/v) Triton X-100, and blocked with 1.25% (w/v) heat-denatured BSA for 30 min at room temperature. Cells were then incubated with an anti-GST monoclonal antibody  at 4°C overnight, followed by incubation with FITC-conjugated goat anti-rabbit IgG (1∶500) at 37°C for another 30 min, and imaged using immunofluorescence confocal microscopy .Three hundred microliter of SLK cell suspensions were seeded on each cover slips(3×105 cells/ml in RPMI1640 with 10% FCS). After incubation for 1 hour at 37°C in a 5% CO2 atmosphere, non-adherent cells were removed by careful aspiration and three washes with RPMI1640. Adherent cells were fixed, stained, and quantitated as describedTo guarantee the same amount of tested protein was coated, anti-GST antibody was adsorbed to polystyrene microplates  by overnight incubation at 4°C in 100 mM carbonate buffer (pH 9.6) at a concentration of 10 µg/ml. The plates were subsequently washed three times with tris-buffered saline+Tween 20 (TBS-T) and then filled with 1.25% BSA-PBS and incubated for 2 h at 37°C. After three washes with TBS-T, 40 µg/ml of GST alone, GST-Tat or the relevant mutant Tat protein was added to the wells and incubated for 1 hour at 37°C. The plate was next washed three times with PBS-T. Then 100 µl of SLK cell suspension was added to each well (3×10The protein coating and cell treatment were similiar as the procedure of cell adhesion except using acid-washed cover slips instead of polystyrene microplates. The adherent cells were then fixed in 4%(v/v) paraformaldehyde for 30 min, permeabilized with 0.1%(v/v) Triton X-100 in PBS for 10 min at room temperature, and treated with blocking buffer (1.25% (w/v) BSA in PBS). Cells were then incubated with monoclonal anti-12G10 (1∶100) directed against the activated-β1 integrin at 4°C overnight, followed by incubation with Cy3-conjugated anti-mouse IgG (1∶1000) at 37°C for another 30 min and imaged using immunofluorescence confocal microscopy. A heparin-saturation integrin clustering assay was conducted using the same procedure except that heparin  was added to the wells and incubated for 1 hour with GST, GST-Tat or the appropriate mutant Tat protein before adding cells.Text S1Supplementary Methods and Results(0.03 MB DOC)Click here for additional data file.Figure S1The representative MS-MS spectrum of GST-Tat. A, The MS-MS spectrum of GST-Tat peptide, LEPWKHPGSQPK, with Xcorr score 2.48; and B, The MS-MS spectrum of GST-Tat peptide, RPPQGSQTHQVSLSK, with Xcorr score 4.03.(0.97 MB TIF)Click here for additional data file.Figure S2The MS-MS spectrum of GST-Tat mutant GST-Tat(G48-R57)A. The peptide ALGISYAAAAAAAAAAPPQGSQTHQVSLSK, with Xcorr score of 5.62, indication of the 48–57 residues substituted by Alanine (The mutated amino acids are underlined).(0.53 MB TIF)Click here for additional data file.Figure S3The MS-MS spectrum of GST-Tat mutant GST-TatKA/R78A. A, The MS-MS spectrum of GST-TatKA/R78A peptide, LEPWAHPGSQPK, with Xcorr score 2.78, indication of the twelfth residue Lysine of Tat substituted by Alanine; B, The MS-MS spectrum of GST-TatKA/R78A peptide, CCFHCQVCFITAALGISYGR, with Xcorr score 5.32, indication of the forty-first residue Lysine of Tat substituted by Alanine; and C, The MS-MS spectrum of GST-TatKA/R78A peptide, QPTSQSAGDPTGPK, with Xcorr score 2.8, indication of the seventy-eighth Arg of Tat substituted by Alanine. The mutated amino acids are underlined.(1.65 MB TIF)Click here for additional data file.Figure S4The MS-MS spectrum of GST-Tat mutant GST-Tat(G48-R57)A/KA/R78A. A, The MS-MS spectrum of GST-Tat(G48-R57)A/KA/R78A peptide, LEPWAHPGSQPK, with Xcorr score 2.9, indication of the twelfth residue Lysine of Tat substituted by Alanine; B, The MS-MS spectrum of GST-Tat(G48-R57)A/KA/R78A peptide, CCFHCQVCFITAALGISYAAAAAAAAAAPPQGSQTHQVSLSK, with Xcorr score 4.83, indication of the forty-first residue Lysine and the 48–57 residues of Tat substituted by Alanine; and C, The MS-MS spectrum of GST-Tat(G48-R57)A/KA/R78A peptide, QPTSQSAGDPTGPKE, with Xcorr score 3.14, indication of the seventy-eighth Arg of Tat substituted by Alanine. The mutated amino acids are underlined.(1.67 MB TIF)Click here for additional data file.


The most efficient and stable reporter activity was observed for combinations of the transposon inverted terminal repeats and one 5’- or two cHS4 core insulators flanking a green fluorescent protein reporter construct, with no detectable silencing over 10 months of continuous cell culture in absence of any selective pressure. In monocytic THP-1 cells, the functional activity of luciferase reporters for NF-κB, Nrf2, or HIF-1α has not decreased over time and was retained following differentiation into macrophages and DCs, as well. These results imply pB as a versatile tool for gene integration in monocytic cells in general, and as a convenient access route to DC-based signaling pathway reporters suitable for high-throughput assays, in particular. Genetically modified hematopoietic progenitors represent an important testing platform for a variety of cell-based therapies, pharmaceuticals, diagnostics and other applications. Stable expression of a transfected gene of interest in the cells is often obstructed by its silencing. DNA transposons offer an attractive non-viral alternative of transgene integration into the host genome, but their broad applicability to leukocytes and other “transgene unfriendly” cells has not been fully demonstrated. Here we assess stability of  The sigter gene ,3, toxicter gene ,5, whichex vivo. Dendritic cells are professional antigen-presenting cells and are key regulators of the inflammatory status in response to the microbiota, environmental stress and pathogen invasions [in vitro from monocytic precursors [Blood-borne cells, such as dendritic cells (DCs) or their monocyte progenitors, represent an attractive resource for developing various human cell-based assays ,7, due tnvasions ,9. Maturecursors . Howeverecursors . An alteecursors ,13. In aecursors  using qPcis- and trans-acting factors, including spatial organization of chromatin, nucleosomal positioning on regulatory sequences, and covalent modifications of DNA nucleotides and histones. Random exogenous polynucleotide integration into the host DNA often leads to the transgene silencing [per se do not provide transgene protection from silencing, while their cargo size limitations and safety concerns have prompted an increase in efforts to develop efficient non-viral gene delivery and protection tools [Stable expression of transgenes in human cells has received a significant effort from both academia and pharma, due to a wide variety of potential applications for genetically modified cells ,16. Reguilencing . To achiilencing . Althougon tools . The taspiggyBac transposon (pB) was initially identified in insects over three decades ago, but has been introduced into the gene technology field only recently [pB transposon has a number of advantages over traditional transfection and viral gene delivery approaches. First, the transgene integration into the target genome is not spontaneous, but is mediated by co-transfected pB transposase (pBase) in a “cut-and-paste” fashion. Second, the insertion sites tend to localize in transcription-active areas rich in CpG islands. Finally, the size of pB transposon cargo gene (over 200 kb [The recently . The pB r 200 kb ) dramaticis-regulatory elements that can block improper gene activation or heterochromatin propagation imposed by remote enhancers and silencers. One of the best studied insulators is chicken hypersensitive site-4 (cHS4) boundary element at the β-globin locus, which acts by recruiting histone acetyl transferases and hindering DNA methylation of the transgene promoter [Insulators are promoter . In addipromoter ,23.pB transposon mediated transfer of insulated reporter gene series into THP-1 cells could provide for stably altered monocytic cell lines capable of further cell differentiation into tissue-specific macrophages or DCs with the reporter activities preserved. To the best of our knowledge, this approach enabled, for the first time, a facile generation of DC-based signaling pathway reporters suitable for high-throughput assays. In this study, we have assessed whether The overall efficiency of stable transgene expression depends on the gene delivery, integration into the host genome, and epigenetic regulation of its promoters due to the positional and silencing/ enhancing effects. Monocyte-derived cells are well equipped for recognition and clearance of foreign DNA , and arepiggyBac transposase (pBase) plasmid and sorted by flow cytometry, the GFP-positive cells have uniformly retained GFP expression and puromycin resistance (Transgenic mouse prostate adenocarcinoma (TRAMP) is a popular model for prostate cancer studies, both in vitro and in vivo ,27. Reposistance . pB transposon inverted terminal repeat (ITR) recognition sites and two core HS4 insulators flanking the GFP reporter/ puromycin resistance sequence , along with the pBase plasmid. All transfects were enriched by fluorescence-activated cell sorting (FACS) in 5 to 6 biweekly sort rounds, until 100% fluorescent, and then left to proliferate for 4 months without any further selection. The flow cytometry analysis is presented in A, which lacks both transposon and insulator sequences. Cells transfected with pSB through pSE, that is, possessing either pB or at least one cHS4 insulator, formed a group with an intermediate fluorescence intensity (median 84 to 107-fold). The strongest median fluorescence (214 to 366-fold) was displayed by cells transfected with vectors pSF through pSH, containing both pB and at least one cHS4. At the end of the 4-month run , transposon only (pSE), or transposon plus one insulator (pSF and pSG), we observed a shift in the cell populations towards 2 to 3-fold decrease in median GFP expression, but no cells with a complete loss of the reporter. In contrast, there was only about 20% decrease in median GFP expression in cells that have the construct H, with both insulators flanking the GFP reporter gene, that were inserted into the chromatin by pB transposition.pB513B-1 contains both sequence . To deteA, pSD, or pSH and then FACS-selected for the uniformity of fluorescence, the resulting stable transfects displayed a GFP expression intensity and stability pattern, in the order pSH>pSD>pSA  have retained the potential of immature phagocytic progenitors, we performed differentiation of the monocyte transfects into mature macrophages or DCs. When THP.R0nF were treated with phorbol 12-myristate 13-acetate (PMA) for 2 days following a standard differentiation protocol, all cells became tightly adherent, with characteristic macrophage morphology . However4 cells/well, and some were treated with the differentiating agents for 2 days in serum-free media. The differentiation media were replaced with an assay medium to adapt for next 16-24 h. Following the adaptation period, specific pathway stimulating agents were added to the monocyte, macrophage and DC reporters for desired periods of time. Then treated and untreated cells were lysed, and fluorescence of GFP in the lysates was measured in a capacity of an internal cell number surrogate, in order to evaluate the cell proliferation/death rate and to normalize the following luminescence data. Finally, the luciferin substrate was added to the lysates, and the luminescence in wells was measured. The assay data are compiled in Next, inducible reporter luciferase activity for each signaling pathway reporter was compared between the monocytes and respective differentiated macrophages and DCs. Aiming at development of practical high-throughput assays, the comparative studies were carried out in a 96-well plate format. According to a protocol which we followed, THP.R0nF were seeded into the inner wells of 96-well plates, at 2×10As follows from 2 and o-phenanthroline, however, the strongest response was detected in the macrophages, 24-fold increase, and DCs (16-fold), also in a dose-dependent manner  and K562. These were tested in presence of the NF-κB, HIF-1α, or Nrf2 activators, and the results are depicted in piggyBac transposon-mediated gene transfer to successfully introduce functional signaling pathway reporters into a monocytic line THP-1 and the monocyte-derived macrophages and dendritic cells. Although transposons have previously been used for gene delivery into hematopoietic stem cells and lymphocytes, this work deals with monocyte-derived cells and thus widens the scope of potential applications for the transposon technology in immunology related areas. For example, stabilization of hypoxia-inducible therapeutic gene expression in tumor-infiltrating macrophages could potentially aid in development of cell-based antitumor therapies [in vitro alternative for environmental allergen assays, such as those that currently use animals for skin or mucosa sensitization [In this study, we employed herapies ,32. Anottization ,33. pB employed in this work, there are two other transposon systems currently being actively developed as potential candidates for therapeutic gene administration into the human genome: Sleeping Beauty, and Tol2. A few publications offer comparisons of the three transposons head-to-head [pB may be well achievable with the other “humanized” transposons, establishing the relative transposon efficiency was not critically significant for the purpose of this work. More importantly, we have opted in favor of a transposon system over virus-based vectors following a failure of the lentivirus-mediated gene transduction to provide for stable expression of a GFP reporter in TRAMP-C2 , cadmium acetate, cobalt chloride, nickel chloride, oltipraz, tert-butylhydroquinone, o-phenanthroline were from Sigma-Aldrich, human IL-1β and murine TNFα were purchased from PeproTech, while CDDO-Me was a gift from TransTech Pharma.Cell differentiation agents human GM-CSF and IL-4 were from Shenandoah Biotech; PMA, ionomycin, lipopolysaccharide  supplemented with 5% fetal bovine serum (Gibco) and 1% (v/v) penicillin/streptomycin cocktail (CellGro). The cells were subcultured 1:10 upon reaching near confluency or when detached. THP-1 and all related transfects were cultured in the RPMI-1640 supplemented with 10% heat-inactivated FBS (HyClone), additional 2 g/L glucose, 1 mM sodium pyruvate, 10 mM HEPES (both from Sigma), and 1% penicillin/streptomycin. The cell density was maintained between 1×10piggyBac transposase expression vector and pB513B-1 transposon vector were purchased from System Biosciences . Super B: Plasmid pB513B-1 was digested with SwaI/EcoRV restriction enzymes, and the smaller fragment was inserted into pSmart_HCKan  using the CloneSmart reaction.pSF: The pB ITRs were obtained by PCR of pB513B-1 with the following primer sets. For 3’ITR: TCGTTAAATCGGATCGAACACGCAGCTAGATTAAC and TTGCGGCCGCGGATCCGATCTCGATATACAGACCGATAA. For 5’ITR : CAATACTGACCATTTAAATCGCTATTTAGAAAGAGAGAG and GGTCAGGTATGATTTACAGCTATGACCATGATTAC. Then pSB was digested with BamHI/ SwaI and both pieces isolated. These four fragments were assembled into pSF in a one-step In-Fusion reaction according to the manufacturer’s (Clontech) protocol. pSA and pSE: The 5’HS4 insulator was excised from pSB and pSF with BstXI/ XbaI, and the gap was In-Fusion-patched with a PCR product of pB513B-1 and the following primers: TTTTTTAGGGCCCATGTAATACGGTTATCCACAGA and TTGAGCGATATCTAGACTGGTATCTTTATAGTCCTGTC. pSC, pSD, pSG, and pSH: The 3’HS4 insulator was obtained by PCR of pB513B-1 with the following primers: ATCGGATCCGCGGCCGGTCTGTATATCGAGGTTTA and CAGATCCTTGCGGCCGCCATAATACTAGTAGGCCTTGG. It was brought into the In-Fusion reaction with NotI digests of pSA, pSB, pSE, and pSF, respectively.pSTGGCAGTACATCTACGTAAACTAGCAAAATAGGCTGTC and CCGCGGATCCGATTTAGAAGGTAGCTAACCAAGTT. Next, P513B-1 was digested with SnaBI/ SwaI. The larger DNA fragment was then brought into the In-Fusion reaction with the reporter PCR product.pTR01F: The NF-κB pathway reporter sequence  has beenNheI/ BglII were assembled into pTR03F in one step following the In-Fusion protocol.pTR03F: Two inserts containing 15-bp overhang sequences for the In-Fusion reaction and total 8 hypoxia response elements  and S9 wpTR05F: In a similar manner, two inserts containing the 15-bp overhang sequences and total 8 antioxidant/electrophile response elements  and S9 wAll constructs were confirmed by DNA sequence analysis.4 cells per well in antibiotic-free RPMI-1640 supplemented with 5% FBS and left to adhere for 6 hours. The cells were then treated with 100 ng of reporter plasmid constructs complexed with TransIT Prostate reagent (Mirus) at 1:2 (μg/ μL) ratio, without the Boost reagent. See pB transposase vector was co-transfected along with pB transposon plasmids. After 16 h, the regular media were added and the cells were left to proliferate for next 48 h. The transfected cells were then treated with a selecting antibiotic (100 μg/ mL G418 or 5 μg/ mL puromycin) for another week, and the surviving cells were expanded for cryopreservation and flow cytometry experiments.TRAMP-C2 and K562 were seeded into wells of a 96-well plate, at 1×105 THP-1 cells in 300 μL RPMI-1640, supplemented with 5% heat-inactivated FBS, were co-treated with 66 ng Super pB transposase plasmid and 200 ng of pB transposon reporter vectors complexed with GeneIn Transfection reagent set  at 1:8 (μg/ μL) ratio. After 16 h, the regular media were added and the cells were left to proliferate for next 48 h. Typical percentage of fluorescent cells at this point was, depending on the vector size, from 0.5 to 5%; see Suspensions of 2×105 TU per well containing 1×104 cells, were carried out according to the manufacturer’s protocol.Tranductions of TRAMP-C2 with Lenti GFP Control (Qiagen), at 1×104 cells/well in 96-well plates. The cells were then treated with 10 ng PMA/well for 3 days, when all cells became adherent and assumed the macrophage morphology. The transformed cells were washed and kept in the RPMI-1640  for a day, before any assays.Suspensions of the original THP-1 or the reporter lines in the differentiation serum-free medium CellGro Complete (Mediatech/ Corning) have been seeded at 2×10The cells were seeded as above, using serum-free OptiMEM I (Gibco/ Life Technologies) as a differentiation medium and then treated with 10 ng GM-CSF and 20 ng IL-4 per well for 16 hours, followed by 2 ng TNFα and 20 ng ionomycin per well . After 22, 100% humidity). TRAMP-C2 and K562-derived reporters were seeded at 1×104 or 2×104 cells/well, respectively, in the culturing medium two days prior treatments. The assay medium was the complete RPMI-1640 less Phenol Red indicator. After the treatments, the reporter cells were washed and lysed in 60 μL of the Luciferase reporter lysing buffer (Promega). The lysates fluorescence was measured at the 482(9)/512(17) nm wavelength(width) setup, followed by addition of the luciferase substrate (Promega) and kinetic luminescence measurements in the wells, with 2-minute intervals, for 30 minutes total. All the measurements were done using a Synergy MX (BioTek) plate reader. The GFP fluorescence values were used for both evaluation of relative cell proliferation and normalization of the reporter luciferase activities in respective wells.In a typical experiment, naïve or differentiated THP reporters seeded in 96-well plates were treated with sensitizers in 100 μL of serum-free CellGro Complete medium (Corning) for specific times in the standard cell culturing conditions . Analysis of the FACS data was performed with help of Summit 5.2 software package.Statistical tests and plots were done using SigmaPlot software, version 11.0.Figure S1Conventional transfections or viral transduction fail to provide stable expression of GFP in TRAMP-C2 cells. TRAMP-C2 were A) transfected with a control pEGFP-C1 plasmid, B) transduced with Cignal LentiGFP, or C) co-transfected with PB513B-1 and pBase. The cells then went through several rounds of fluorescence activated cell sorting in two-week intervals, by selecting 5% of the brightest (R4 cut on the histograms) cells for further proliferation. The respective fluorescence histograms are positioned in the columns and the sort rounds – in rows, from top to bottom. The cytometer settings were not uniformly calibrated on different days in this experiment. The fluorescence profiles of transfected/transduced TRAMP-C2 cells are shown in solid blue, while auto-fluorescence profiles of non-transfected cells are overlayed as contour lines. (EPS)Click here for additional data file.Figure S2A representative view of the fluorescence “mosaic” distribution in FAC-sorted (6 rounds) DU145 cells transfected with a control GFP plasmid. (EPS)Click here for additional data file.Figure S3pB-based, insulated reporters are stably expressed in K562 line. K562 cells were co-transfected with pB transposase and pSA, pSD, or pSH reporter plasmids. The transfects went through several rounds of FAC sorting, until the maximum fluorescence has been achieved (the upper histogram in each pair). The sorted cells were then let to proliferate without any selection and were subcultured twice a week. The lower histogram in each pair represents FACS analysis of the cells at the end of the 3-month term. The FACS instrument settings were uniformly calibrated against Sphero Rainbow Beads. Median fluorescence intensities were normalized for the non-transfected control K562 cell autofluorescence. (EPS)Click here for additional data file.Figure S4Schematic representation of the pathway reporter constructs, which are flanked by pB and cHS4 sequences as in H . The transcription response element, minimal CMV promoter, and firefly luciferase encoding sequences were inserted into p513B-1, resulting in pTR01F, pTR03F, and pTR05F plasmids. Sequences of TREs for HIF-1α and Nrf2 are given with essential nucleotides capitalized. TREs for NF-κB were cloned from pGL4.32. (EPS)Click here for additional data file.Figure S5NF-κB reporter activity in TC2.R01F. The cells were treated with proinflammatory agents, in triplicates, for 6 hours, then lysed. A. Fluorescence in the lysates was normalized against the control. B. Luciferase activities in the lysates were normalized against the respective fluorescence values and then against the control. The bars and error bars are normalized fluorescence or luminescence means ± SD, n = 3; statistical analysis: pairwise comparisons by an unpaired t-test. Statistical significance *: the lowest dose treatment compared to control; in all cases, P<0.001. (EPS)Click here for additional data file.Figure S6HIF-1α reporter activity in TC2.R03F. The cells were treated with hypoxia mimicking agents, in triplicates, for 15 hours, then lysed. A. Fluorescence in the lysates was normalized against the control. B. Luciferase activities in the lysates were normalized against the respective fluorescence values and then against the control. The bars and error bars are normalized fluorescence or luminescence means ± SD, n = 3; statistical analysis: pairwise comparisons by an unpaired t-test. Statistical significance *: the lowest dose treatment compared to control. (EPS)Click here for additional data file.Figure S7Nrf2 reporter activity in TC2.R05F. TC2.R05F cells were treated with electrophilic agents, in triplicates, for 14 hours, then lysed. A. Fluorescence in the lysates was normalized against the control. B. Luciferase activities in the lysates were normalized against the respective fluorescence values and then against the control. The bars and error bars are normalized fluorescence or luminescence means ± SD, n = 3; statistical analysis: pairwise comparisons by an unpaired t-test. Statistical significance *: the lowest dose treatment compared to control. (EPS)Click here for additional data file.Figure S8NF-κB reporter activity in K562.R01F. The cells were treated with proinflammatory agents, in triplicates, for 6 hours, then lysed. A. Fluorescence in the lysates was normalized against the control. B. Luciferase activities in the lysates were normalized against the respective fluorescence values and then against the control. The bars and error bars are normalized fluorescence or luminescence means ± SD, n = 3; statistical analysis: pairwise comparisons by an unpaired t-test. Statistical significance *: the lowest dose treatment compared to control; in all cases, P<0.05. (EPS)Click here for additional data file.Figure S9Octamer transcription response element sequences for the In-Fusion mediated construction of pTR03F and pTR05F vectors, respectively.(EPS)Click here for additional data file.Table S1Efficiencies (%) of transient transfections of the cell lines after 48 hours.(DOCX)Click here for additional data file.