MYCNAMP NB4 70, a novel miRNA-like sequence resulting from a duplication event

<p><b>Copyright information:</b></p><p>Taken from "New miRNAs cloned from neuroblastoma"</p><p>http://www.biomedcentral.com/1471-2164/9/52</p><p>BMC Genomics 2008;9():52-52.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254388.</p><p></p> Short sequences flanking MYCNAMP_NB4_70 were extracted from the human genome, as well as homologuous sequences from chimpanzee, macaque, mouse, rat and dog genomes and aligned. The bar indicates the MYCNAMP_NB4_70 sequence. The duplication is marked by a box

Predicted secondary structures of the putative novel human miRNA precursors

<p><b>Copyright information:</b></p><p>Taken from "New miRNAs cloned from neuroblastoma"</p><p>http://www.biomedcentral.com/1471-2164/9/52</p><p>BMC Genomics 2008;9():52-52.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254388.</p><p></p> Human genomic sequences upstream and downstream of the novel miRNAs were folded with the computer program RNAfold. Grey areas represent the cloned miRNA. a) miRNA with canonical hairpin. b) borderline structures

SERpredict: Detection of tissue- or tumor-specific isoforms generated through exonization of transposable elements-2

<p><b>Copyright information:</b></p><p>Taken from "SERpredict: Detection of tissue- or tumor-specific isoforms generated through exonization of transposable elements"</p><p>http://www.biomedcentral.com/1471-2156/8/78</p><p>BMC Genetics 2007;8():78-78.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2194731.</p><p></p> through the exonization of an Alu element

SERpredict: Detection of tissue- or tumor-specific isoforms generated through exonization of transposable elements-0

<p><b>Copyright information:</b></p><p>Taken from "SERpredict: Detection of tissue- or tumor-specific isoforms generated through exonization of transposable elements"</p><p>http://www.biomedcentral.com/1471-2156/8/78</p><p>BMC Genetics 2007;8():78-78.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2194731.</p><p></p>or the different tissues

Modifications of FFV <i>bel2</i> with OVA do not significantly influence viral protein levels or infectivity.

<p>A) Schematic representation of epitope replacements in Bet. Original protein sequences are shown in black; modifications in red. Epitope sequences are underlined. B) Cell lysates and C) enriched culture supernatants of 293T cells transfected with modified and wild-type proviruses were analyzed by immunoblotting. Cells and supernatants were harvested 2 d post-transfection and probed using polyclonal sera against the Gag matrix and Env transmembrane domains. Detection of Env and Gag in the particulate fraction represents specifically released virus particles. The Gag precursor (p52), cleaved mature Gag (p48), Env precursor (gp130Env), mature TM (gp48TM) and a cell lysate-associated transmembrane isoform (TM^CL) are indicated by arrows. Proper protein loading of cell lysates was determined by probing for β-actin. D) CrFK and E) KE-R cells were infected with virus-containing supernatants harvested from 293T cells transfected with modified and wild-type proviruses. Supernatants were passaged in uninfected cells every two days. Viral titers were determined by titration on FeFAB cells. Cells were mock-infected with supernatant from pcDNA-transfected cells as a negative control. Titers are presented as mean values of three independent experiments. Error bars represent standard deviation of mean values.</p

Target cells infected by FFV expressing Bet-SIINFEKL vectors stimulate SIINFEKL-specific CTLs.

<p>For infection by supernatant transfer, 293T cells were transfected with pCF-7, pCF-Bet-Ova8, pCF-Bet-Ova12, pCF-Bet-Ova16, pCF-Gag-Ova, or pcDNA. Two d p.t., culture supernatants were cleared and applied onto EL4 cells. For infection by co-culture, 293T cells transfected with pCF-7, pCF-Bet-Ova8, or pcDNA were cultured together with EL4 cells 2 d p.t. Thereafter, cells were harvested and co-cultured with OVA-specific CTLs in ELISpot assays. Plates were probed after 24 h of co-cultivation for the presence of secreted IFNγ. The stable OVA-expressing cell line EG7 served as positive control (black bar). Untransfected EL4, EL4 transfected with pCF7 or with pcDNA were used as negative controls (white bars). Results are shown as number of IFNγ spots per 5000 CTLs/well. Values represent a single experiment performed in triplicate. * represents a p-value of less than 0.05 when compared to pCF-7; **, p-value < 0.01; ***, p-value < 0.001.</p

Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy

<div><p>The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated <i>in vitro</i> for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.</p></div

Activation of SIINFEKL-specific CTL by pCF-Bet-Ova8 infected EL4 cells is not due to external peptide loading.

<p>HEK293T cells were transiently transfected with pCF-7 or pCF-Bet-Ova8. After 2 d, cell culture supernatants were harvested, cleared, and passed through a 100-kDa centrifugal filter. A) The filtrate (dotted bars) and retentate (striped bars) fractions were applied onto EL4 cells. After 4 d, EL4 cells were harvested and co-cultured with OVA-specific CTLs in an ELISpot assay. Plates were probed after 24 h of co-cultivation for the presence of secreted IFNγ. EG7 cells were used as positive control (black bar). EL4 cells, either untreated or infected with wild-type FFV, and corresponding filtrate fractions were used as negative controls (white bars). Results are shown as number of IFNγ spots per 12500 CTLs/well. ** represents a p-value of less than 0.01 when compared to pCF-7. B) Viral titers were determined for the retentate and filtrate fractions, confirming absence of viral particles in the filtrates.</p

Proper processing and presentation of the OVA-specific H2-K^b-restricted CTL epitope SIINFEKL from the C-terminus of Bet.

<p>EL4 cells nucleofected with different pmaxBet-Ova epitope expression constructs present the H2-K^b-restricted CTL epitope SIINFEKL on the cell surface, stimulating IFNγ release by SIINFEKL-specific CTLs. Untransfected EL4 cells or EL4 cells transfected with, pmaxGFP, pmaxBet, and pcDNA served as negative controls. The OVA expressing EL4-derived transfectant clone EG7 as well as addition of concanavilin A were used as positive controls. Values represent a single experiment performed in triplicate. *** represents a p-value of less than 0.001 when compared to pmaxBet.</p

Most modifications in structural proteins are detrimental to viral protein steady state levels and infectivity.

<p>(A) Schematic representation of the FFV genome with the long terminal repeats (LTR) and the open reading frames shown as boxes and the promoters in the 5’ LTR and the internal promoter as broken arrows pointing into the direction of transcription. The epitope replacements in Env (B) and Gag (C) are shown below with original protein sequences in black and modifications in red. Epitope sequences are underlined. CrFK (D) and KE-R (E) cells were infected with virus-containing supernatants harvested from 293T cells transfected with wild-type and modified proviruses. Supernatants were serially passaged onto uninfected cells every two days. Viral titers were determined by titration on FeFAB cells. Cells were mock-infected with supernatant from pcDNA-transfected cells as a negative control. Titers are presented as mean values of three independent experiments. Error bars represent standard deviation of mean values.</p