Study on Flake Formation Behavior and Its Influence Factors in Cr5 Steel.

A flake is a crack that is induced by trapped hydrogen within steel. To study its formation mechanism, previous studies mostly focused on the formation process and magnitude of hydrogen pressure in hydrogen traps such as cavities and cracks. However, according to recent studies, the hydrogen leads to the decline of the mechanical properties of steel, which is known as hydrogen embrittlement, is another reason for flake formation. In addition, the phenomenon of stress induced hydrogen uphill diffusion should not be neglected. All of the three behaviors are at work simultaneously. In order to further explore the formation mechanism of flakes in steel, the process of flake initiation and growth were studied with the following three coupling factors: trap hydrogen pressure, hydrogen embrittlement, and stress induced hydrogen re-distribution. The analysis model was established using the finite element method, and a crack whose radius is 0.5 mm was set in its center. The cohesive method and Bilinear Traction Separate Law (BTSL) were used to address the coupling effect. The results show that trap hydrogen pressure is the main driving force for flake formation. After the high hydrogen pressure was generated around the trap, a stress field formed. In addition, the trap is the center of stress concentration. Then, hydrogen is concentrated in a distribution around this trap, and most of the steel mechanical properties are reduced. The trap size is a key factor for defining the critical hydrogen content for flake formation and propagation. However, when the trap size exceeds the specified value, the critical hydrogen content does not change any more. As for the crack whose radius is 0.5 mm, the critical hydrogen content of Cr5VMo steel is 2.2 ppm, which is much closer to the maximum safe hydrogen concentration of 2.0 ppm used in China. The work presented in this article increases our understanding of flake formation and propagation mechanisms in steel.

Ubr3, a Novel Modulator of Hh Signaling Affects the Degradation of Costal-2 and Kif7 through Poly-ubiquitination.

Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues.

PI(4)P Promotes Phosphorylation and Conformational Change of Smoothened through Interaction with Its C-terminal Tail.

In Hedgehog (Hh) signaling, binding of Hh to the Patched-Interference Hh (Ptc-Ihog) receptor complex relieves Ptc inhibition on Smoothened (Smo). A longstanding question is how Ptc inhibits Smo and how such inhibition is relieved by Hh stimulation. In this study, we found that Hh elevates production of phosphatidylinositol 4-phosphate (PI(4)P). Increased levels of PI(4)P promote, whereas decreased levels of PI(4)P inhibit, Hh signaling activity. We further found that PI(4)P directly binds Smo through an arginine motif, which then triggers Smo phosphorylation and activation. Moreover, we identified the pleckstrin homology (PH) domain of G protein-coupled receptor kinase 2 (Gprk2) as an essential component for enriching PI(4)P and facilitating Smo activation. PI(4)P also binds mouse Smo (mSmo) and promotes its phosphorylation and ciliary accumulation. Finally, Hh treatment increases the interaction between Smo and PI(4)P but decreases the interaction between Ptc and PI(4)P, indicating that, in addition to promoting PI(4)P production, Hh regulates the pool of PI(4)P associated with Ptc and Smo.

Prostate cancer-specific and potent antitumor effect of a DD3-controlled oncolytic virus harboring the PTEN gene.

Prostate cancer is a major health problem for men in Western societies. Here we report a Prostate Cancer-Specific Targeting Gene-Viro-Therapy (CTGVT-PCa), in which PTEN was inserted into a DD3-controlled oncolytic viral vector (OV) to form Ad.DD3.E1A.E1B(Δ55)-(PTEN) or, briefly, Ad.DD3.D55-PTEN. The woodchuck post-transcriptional element (WPRE) was also introduced at the downstream of the E1A coding sequence, resulting in much higher expression of the E1A gene. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical bio-diagnostic marker. PTEN is frequently inactivated in primary prostate cancers, which is crucial for prostate cancer progression. Therefore, the Ad.DD3.D55-PTEN has prostate cancer specific and potent antitumor effect. The tumor growth rate was almost completely inhibited with the final tumor volume after Ad.DD3.D55-PTEN treatment less than the initial volume at the beginning of Ad.DD3.D55-PTEN treatment, which shows the powerful antitumor effect of Ad.DD3.D55-PTEN on prostate cancer tumor growth. The CTGVT-PCa construct reported here killed all of the prostate cancer cell lines tested, such as DU145, 22RV1 and CL1, but had a reduced or no killing effect on all the non-prostate cancer cell lines tested. The mechanism of action of Ad.DD3.D55-PTEN was due to the induction of apoptosis, as detected by TUNEL assays and flow cytometry. The apoptosis was mediated by mitochondria-dependent and -independent pathways, as determined by caspase assays and mitochondrial membrane potential.

Suppression of colorectal tumor growth by regulated survivin targeting.

A major goal in cancer gene therapy is to develop efficient gene transfer protocols that allow tissue-specific and tightly regulated expression of therapeutic genes. The ideal vector should efficiently transduce cancer cells with minimal toxicity on normal tissues and persistently express foreign genes. One of the most promising regulatory systems is the mifepristone/RU486-regulated system, which has much lower basal transcriptional activity and high inducibility. In this work, we modified this system by incorporating a cancer-specific promoter, the human telomerase reverse transcriptase (hTERT) promoter. By utilizing hTERT promoter to control the regulator, RU486 could specifically induce the expression of foreign genes in cancer cells but not in normal cells. In the context of this system, a dominant negative mutant of survivin (surDN) was controllably expressed in colorectal tumor cells. The surDN expression induced by RU486 showed a dosage- and time-dependent pattern. Regulated expression of surDN caused caspase-dependent apoptosis in colorectal tumor cells but had little effect on normal cells. Analysis of cell viability showed that RU486-induced expression of surDN suppressed colorectal tumor cell growth and had synergic effect in combination with chemotherapeutic agents. The potential of this system in cancer therapy was evaluated in experimental animals. Tumor xenograft models were established in nude mice with colorectal tumor cells, and RU486 was intraperitoneally administered. The results showed that conditional expression of surDN efficiently inhibited tumor growth in vivo and prolonged the life of tumor-burdened mice. Synergized with the chemotherapeutic drug cisplatin, regulated surDN expression completely suppressed tumor growth. These results indicated that this modified RU486-regulated system could be useful in cancer-targeting therapy.

A survivin-mediated oncolytic adenovirus induces non-apoptotic cell death in lung cancer cells and shows antitumoral potential in vivo.

BACKGROUND: Conditionally replicating adenoviruses or oncolytic adenoviruses, which can replicate selectively in tumor cells and kill them, represent an innovative class of promising cancer therapeutics. Survivin is the smallest member of the inhibitor of apoptosis (IAP) family, which is transcriptionally upregulated exclusively in most malignant tissues but not in normal tissues. It has been reported that activity of the survivin promoter is tumor-specific, which makes the survivin promoter a good candidate to construct oncolytic viral vectors. METHODS: A luciferase reporter assay was used to determine the activity of the survivin promoter in tumor and normal cells. An oncolytic adenovirus (Ad.SP/E1A) was generated by homologous recombination. The oncolytic efficacy of Ad.SP/E1A was evaluated in cell lines and in a human lung xenograft tumor mouse model. RESULTS: Survivin expression was highly upregulated in tumor cells both at the protein and mRNA level. The luciferase reporter assay showed that survivin promoter activity is tumor-specific. Ad.SP/E1A expressed E1A selectively in tumor cells and induced cytotoxicity, but not in normal cells. Moreover, in animal experiments, intratumoral administration of Ad.SP/E1A significantly suppressed the growth of xenograft tumors. Further investigation showed that Ad.SP/E1A induced cell death by an apoptosis-independent pathway. CONCLUSIONS: Ad.SP/E1A could be a potent therapeutic agent for cancer gene therapy. The investigation of the mechanisms of oncolytic virus-induced cell death in this work will shed light on the construction of more powerful vectors for cancer therapy.

Herpes virus proteins ICP0 and BICP0 can activate NF-kappaB by catalyzing IkappaBalpha ubiquitination.

The immediate early proteins ICP0 and BICP0 from Herpes virus are promiscuous activators of both viral and cellular genes and play a critical role in virus life cycle. Here we report that ICP0 and BICP0 could induce NF-kappaB translocation from cytoplasm into nucleus and strongly activate NF-kappaB responsive genes specifically. This process was dependent on the RING domain of both proteins. In addition, ICP0 interacted specifically with IkappaBalpha and its activating effect was attenuated by Ubch5A(C85A) and MG132, but not by IkappaBalpha(S32A/S36A). Remarkably, IkappaBalpha was poly-ubiquitinated by both ICP0 and BICP0, in vitro and in vivo. These data indicate that ICP0 and BICP0, functioning as ubiquitin ligases, are bona fide activators of NF-kappaB signaling pathway. Our study identifies a new way ICP0 and BICP0 explore to regulate gene expression.