Comparison of the activation of truncated forms of PTPRZ1 luciferase reporter by HIF-1α and HIF-2α and their degradation-resistant forms.

<p>(A) Hep3B or (B) HEK293T cells were co-transfected with 300 ng of each PTPRZ1 promoter and 50 ng of an internal β-gal control plasmid in the presence of 250 ng of an expression plasmid encoding HIF-1α, drHIF-1α, HIF-2α, drHIF-2α, or pcDNA3.1control. Results are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009641#pone-0009641-g002" target="_blank">Figure 2</a>.</p

HIF-1 and HIF-2 bind to PTPRZ1 oligonucleotide probes containing HRE4.

<p>DIG-labeled synthetic oligonucleotide containing HRE4 was incubated with nuclear extracts (NE) from normoxic (N), 16 hour hypoxic (H) or HIF-transfected (HIF-1α or HIF-2α) HEK293T cells and analyzed on a non-denaturing polyacrylamide gel. Where indicated, unlabeled wild type (WT) or mutant (Mut) oligonucleotides (at 50x and 100x of the labeled probe) were added to the binding reaction. Protein-DNA complexes were separated, blotted to a nylon membrane, and probed with anti-digoxigenin antibody conjugated to alkaline phosphatase. Comp denotes unlabeled probe used for binding competition. The sequence of the probe and WT and Mut competing oligonucleotides used is shown at the bottom. WT HRE4 sequence is underlined, and nucleotide changes in the Mut sequence are shown in bold. The positions of the DIG-labeled HIF complexes and free probe are indicated with arrows.</p

siRNA to ELK1 inhibits PRPRZ1 activation.

<p>(A) Immunoblotting analysis for ELK1, β-Actin, and GAPDH in HEK293T cells transfected with ELK1 scrambled siRNA (60 nM), β-Actin siRNA (60 nM), or ELK1 siRNA (60 nM). (B) ELK1 mRNA as determined by quantitative RT-PCR from HEK293T cells 48 hours after transfection with ELK1 scrambled siRNA, β-Actin siRNA, or ELK1 siRNA. (C) β-Actin mRNA under similar conditions. (D) PTPRZ1-250 promoter activity in HEK293T cells following transfection with HIF-2α plasmid alone or HIF-2α plasmid and either scrambled siRNA, or ELK1 siRNA. Data is presented as fold induction over vector control after normalization to β-gal. Bars represent mean and standard deviation of 3 determinations.</p

Role of EBS4 in the activation of PTPRZ1 promoter by HIF-2α.

<p>(A) Effects of EBS4 or EBS5 deletion on the response of the PTPRZ1 promoter to HIF-2α. HEK293T cells were co-transfected with 300 ng of PTPRZ1-250WT, EBS4D, or EBS5D promoters and 50 ng of a internal β-gal control plasmid in the presence of 250 ng of an expression plasmid encoding HIF-1α, HIF-2α or pcDNA3.1 empty control vector. Results are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009641#pone-0009641-g002" target="_blank">Fig 2</a>. (B). Binding of HIF-2α and HIF-1α to the PTPRZ1 promoter in the region near EBS4, EBS5, HRE4, and HRE5 <i>in vivo</i>. The chromatin immunoprecipitation assay was performed with HEK293T cells transfected with HIF-1α or HIF-2α respectively. Pre-cleared chromatin was immunoprecipitated with anti-HIF-2α or anti-HIF-1α antibody or normal rabbit IgG. After reversal of cross-linking, the DNA was analyzed by PCR. The primer set for PCR were designed to cover the EBS4, EBS5, HRE4, and HRE5 sites (C) Binding of ELK1 to the PTPRZ1 promoter. Experiment performed as in 7B except that the HEK293T cells were transfected with an Elk-1 expression vector and anti-ELK1 antibody or normal rabbit IgG was utilized.</p

PTPRZ1 luciferase promoter constructs showing the location of the potential hypoxia response elements (HRE) and Ets binding sequences (EBS).

<p>Each HRE is denoted as a square and each EBS as a plus sign. The TATA box and the ATG start site are indicated in the PTPRZ1-250 promoter. Each promoter construct extends 57 bp into the PTPRZ1 coding region prior to the luciferase (LUC) sequence except for the PTPRZ1-250 promoter, which stops at the ATG. The HRE consensus sequences and direction of each HRE are also indicated. The core HRE sequences are: HRE1, CCGTG; HRE2, CACGC; HRE3, CACGC; HRE4, CACGCACG; HRE5, CACGG.</p

Semantics-guided generative diffusion model with a 3DMM model condition for face swapping

Face swapping is a technique that replaces a face in a target media with another face of a different identity from a source face image. Currently, research on the effective utilisation of prior knowledge and semantic guidance for photo-realistic face swapping remains limited, despite the impressive synthesis quality achieved by recent generative models. In this paper, we propose a novel conditional Denoising Diffusion Probabilistic Model (DDPM) enforced by a two-level face prior guidance. Specifically, it includes (i) an image-level condition generated by a 3D Morphable Model (3DMM), and (ii) a high-semantic level guidance driven by information extracted from several pre-trained attribute classifiers, for high-quality face image synthesis. Although swapped face image from 3DMM does not achieve photo-realistic quality on its own, it provides a strong image-level prior, in parallel with high-level face semantics, to guide the DDPM for high fidelity image generation. The experimental results demonstrate that our method outperforms state-of-the-art face swapping methods on benchmark datasets in terms of its synthesis quality, and capability to preserve the target face attributes and swap the source face identity.</p

Semantics-guided generative diffusion model with a 3DMM model condition for face swapping

Face swapping is a technique that replaces a face in a target media with another face of a different identity from a source face image. Currently, research on the effective utilisation of prior knowledge and semantic guidance for photo-realistic face swapping remains limited, despite the impressive synthesis quality achieved by recent generative models. In this paper, we propose a novel conditional Denoising Diffusion Probabilistic Model (DDPM) enforced by a two-level face prior guidance. Specifically, it includes (i) an image-level condition generated by a 3D Morphable Model (3DMM), and (ii) a high-semantic level guidance driven by information extracted from several pre-trained attribute classifiers, for high-quality face image synthesis. Although swapped face image from 3DMM does not achieve photo-realistic quality on its own, it provides a strong image-level prior, in parallel with high-level face semantics, to guide the DDPM for high fidelity image generation. The experimental results demonstrate that our method outperforms state-of-the-art face swapping methods on benchmark datasets in terms of its synthesis quality, and capability to preserve the target face attributes and swap the source face identity.</p

Camouflage generative adversarial network: Coverless full-image-to-image hiding

Image hiding, one of the most important data hiding techniques, is widely used to enhance cybersecurity when transmitting multimedia data. In recent years, deep learning based image hiding algorithms have been designed to improve the embedding capacity whilst maintaining sufficient imperceptibility to malicious eavesdroppers. These methods can hide a full-size secret image into a cover image, thus allowing full-image-to image hiding. However, these methods suffer from a trade off challenge to balance the possibility of detection from the container image against the recovery quality of secret image. In this paper, we propose Camouflage Generative Adversarial Network (Cam-GAN), a novel two-stage coverless full-image to-image hiding method named, to tackle this problem. Our method offers a hiding solution through image synthesis to avoid using a modified cover image as the image hiding container and thus enhancing both image hiding imperceptibility and recovery quality of secret images. Our experimental results demonstrate that Cam-GAN outperforms state-of-the-art full-image-to-image hiding algorithms on both aspects

Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses

<div><p>Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi’s sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman’s disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited <i>in vitro</i> and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome, thus thwarting key cellular defense mechanisms.</p></div

Peptides containing LANA caspase cleavage sites inhibit caspase activity, decrease PARP cleavage and increase cell viability.

<p>Caspase activity assays were performed as described in Materials and Methods. Peptides containing the N-terminal caspase cleavage site of LANA (LP-Nterm) or the C-terminal caspase cleavage site of LANA (LP-Cterm) were dissolved in PBS and tested as potential caspase inhibitors at a concentration equal to the commercial caspase-1 substrate (Ac-YEVD-pNa, 200 μM, Sigma) or caspase-3 substrate (Ac-DEVD-pNa, 200 μM, Sigma). PBS was used as the negative control. (A) Effect of LP-Nterm and LP-Cterm on caspase-1 activity. (B) Effect of LP-Nterm and LP-Cterm on caspase-3 activity. In both A and B, the linear caspase activity rate (rfu/min) is plotted with the assay run in triplicate in two separate experiments with one representative experiment shown. Note: The increase in activity of caspase-1 by the LP-Nterm may be due to the presence of a cysteine in this peptide which could protect loss of activity of the enzyme due to oxidation over time. (C) HEP3B cells were pre-treated with DMSO, ZVAD, Tat-LP-Nterm (T-LPN) or Tat-LP-Cterm (T-LPC) peptide for two hrs and then exposed to DMSO alone (control) or etoposide for 16 hrs at 50 μm to induce apoptosis and PARP cleavage. Cell lysates were analyzed by western blot for cleaved PARP and β-actin (loading control). The percent inhibition of PARP cleavage as determined using the LiCor system is indicated below the blot. The immunoblot shown is a representative of 3 different experiments with Tat-LP-Nterm and Tat-LP-Cterm at 50 μM (Tat-LP-Cterm at 100 μM done once). (D) Cell viability following pretreatment with DMSO, ZVAD or T-LPC followed by DMSO or etoposide treatment. Cells were pretreated with DMSO (control), Tat-LP-Cterm or ZVAD (50 μM) for two hrs followed by etoposide treatment at 50 μM. To assess cell viability total cellular protein obtained from the adherent cells following 3 PBS washes was determined by BCA assay. The results shown are the average of 4 independent experiments. ** P< 0.01, * P< 0.05, for two tailed Student’s t-test.</p