IPS-1 differentially induces TRAIL,BCL2, BIRC3 and PRKCE in type I interferons-dependent and -independent anticancer activity

RIG-I-like receptors are the key cytosolic sensors for RNA viruses and induce the production of type I interferons (IFN) and pro-inflammatory cytokines through a sole adaptor IFN-β promoter stimulator-1 (IPS-1) (also known as Cardif, MAVS and VISA) in antiviral innate immunity. These sensors also have a pivotal role in anticancer activity through induction of apoptosis. However, the mechanism for their anticancer activity is poorly understood. Here, we show that anticancer vaccine adjuvant, PolyIC (primarily sensed by MDA5) and the oncolytic virus, Newcastle disease virus (NDV) (sensed by RIG-I), induce anticancer activity. The ectopic expression of IPS-1 into type I IFN-responsive and non-responsive cancer cells induces anticancer activity. PolyIC transfection and NDV infection upregulate pro-apoptotic gene TRAIL and downregulate the anti-apoptotic genes BCL2, BIRC3 and PRKCE. Furthermore, stable knockdown of IPS-1, IRF3 or IRF7 in IFN-non-responsive cancer cells show reduced anticancer activity by suppressing apoptosis via TRAIL and anti-apoptotic genes. Collectively, our study shows that IPS-1 induces anticancer activity through upregulation of pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE via IRF3 and IRF7 in type I IFN-dependent and -independent manners.The primary protection of the host from various pathogens is ensured by the innate immune system, which consists of families of sensors such as the Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors. These sensors recognize the diverse range of pathogens in various cellular compartments and lead to the activation of innate immunity, including the production of various cytokines that create an anti-pathogenic environment to limit the pathogen. RLRs are cytosolic sensors that recognize the viral RNA and recruit an adaptor, Interferon (IFN)-β promoter stimulator-1 (IPS-1), also known as CARDIF, MAVS or VISA. IPS-1, a protein that contains a caspase activation and -recruitment domain (CARD), is localized to the mitochondria for its antiviral function.^{1, 2, 3, 4} Mice lacking IPS-1 show severely impaired antiviral innate immunity.⁵ The RLRs/IPS-1 signaling axis activates a cascade of signals that predominantly induces the production of the type I IFN and pro-inflammatory cytokines through IRFs and NF-κB, respectively, to establish an antiviral state.In addition to the pivotal role that host immunity has against numerous pathogen challenges, it is crucial in immune surveillance against altered-self cells. Immune mediators such as cytokines, chemokines and type I IFN initiate a complex network of signals to induce an anti-tumor state by triggering various biochemical processes such as cell cycle arrest and apoptosis. Additionally, these immune mediators facilitate cytotoxicity to the tumor cells through the recruitment of immunocompetent cells. The cytotoxic activity is mediated through the upregulation of pro-apoptotic genes and the downregulation of anti-apoptotic genes. These changes are critical for cancer cell death.⁶ Various innate and adaptive cytokines are used for treatment of several types of cancer.^{7, 8} The type I IFN are essential for antiviral immunity and induce pleiotropic effects such as the inhibition of malignant growth and apoptosis of altered-self cells.In addition, pathogen-associated molecular patterns such as polyinosinic:polycytidylic acid (polyIC), a synthetic analog of double-stranded RNA and viruses known as oncolytic viruses such as Vesicular stomatitis virus, Newcastle disease virus (NDV) and Sendai virus induce anticancer activity.⁹ However, the molecular mechanisms for these agents are poorly understood.Here, we showed that treatment of cancer cells with polyIC transfection or NDV infection initiates RIG-I- and MDA5-dependent anticancer activity through recruitment of an adaptor, IPS-1. Using IFN α/β receptor1 (IFNAR1)-sufficient and IFNAR1-deficient cancer cells, we showed that these anticancer activities require the RLR signaling pathway. However, type I IFN are dispensable for the anticancer activity. The RLR pathway induces anticancer activity through the selective induction of cell death or apoptosis via upregulation of the pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE. These changes lead to post-translational activation of caspases −3 and −9 and PARP-1 in cancer cells. Furthermore, our study reveals that IFN regulatory factors (IRF)3 and IRF7 are indispensable for the RLR-mediated anticancer activity.     

Configuration of anchorage holes affects fixation of the acetabular component in cemented total hip replacement--a finite element study

Our survey of current practice among UK orthopaedic surgeons shows wide variations in fixation techniques. The aim of this study, is to investigate the effect of drilling different configurations of anchorage holes in the acetabulum on implant stability. To avoid variables that could incur during in vitro testing, we used commercially available COSMOS finite element analysis package to investigate the stress distributions, deformations, and strains on the cement mantle when drilling three large anchorage holes and six smaller ones, with straight and rounded cement pegs. The results, which are in line with our in vitro studies on simulated reconstructed acetabulae, indicate better stability of the acetabular component when three larger holes than six smaller holes are drilled and when the necks of the anchorage holes are rounded. The longevity of total hip replacements could be improved by drilling three large anchorage holes, rather than many smaller ones, as initially proposed by Charnley.     

TAP score: torsion angle propensity normalization applied to local protein structure evaluation

Background  

Experimentally determined protein structures may contain errors and require validation. Conformational criteria based on the Ramachandran plot are mainly used to distinguish bet ween distorted and adequately refined models. While the readily available criteria are sufficient to detect totally wrong structures, establishing the more subtle differences between plausible structures remains more challenging.     

Role of nanotechnology in agriculture with special reference to management of insect pests

Nanotechnology is a promising field of interdisciplinary research. It opens up a wide array of opportunities in various fields like medicine, pharmaceuticals, electronics and agriculture. The potential uses and benefits of nanotechnology are enormous. These include insect pests management through the formulations of nanomaterials-based pesticides and insecticides, enhancement of agricultural productivity using bio-conjugated nanoparticles (encapsulation) for slow release of nutrients and water, nanoparticle-mediated gene or DNA transfer in plants for the development of insect pest-resistant varieties and use of nanomaterials for preparation of different kind of biosensors, which would be useful in remote sensing devices required for precision farming. Traditional strategies like integrated pest management used in agriculture are insufficient, and application of chemical pesticides like DDT have adverse effects on animals and human beings apart from the decline in soil fertility. Therefore, nanotechnology would provide green and efficient alternatives for the management of insect pests in agriculture without harming the nature. This review is focused on traditional strategies used for the management of insect pests, limitations of use of chemical pesticides and potential of nanomaterials in insect pest management as modern approaches of nanotechnology.     

Monitoring Redox Conditions with Flow-Based and Fiber Optic Sensors Based on Redox Indicators: Application to Reductive Dehalogenation in a Bioaugmented Soil Column

Redox indicators were employed to monitor redox status in a bioaugmented, sediment-packed column during the dechlorination of tetrachloroethene (PCE) to ethene (ETH). The speciation of the indicators thionine and cresyl violet, immobilized on transparent films, was spectrometrically monitored with a flow sensor based on circulating the column solution through a specially constructed flow cell placed in a conventional spectrometer. A fiber optic redox probe based on immobilized azure C was constructed. A 75-cm column with 4 sets of ports along the column axis at regular intervals was constructed and packed with aquifer material. These ports enabled sampling to determine concentrations of chlorinated ethene species and allowed for in situ or non-invasive monitoring of redox conditions with negligible O ₂ contamination. The flow sensor and fiber optic sensor showed similar responses to redox conditions in the column. After 60 days, complete conversion of PCE to ETH occurred by the end of the column and the redox level indicated by the indicators was consistent throughout the column. Significant formation of vinyl chloride or ETH was observed only after significant reduction of cresyl violet.     

The Missing Link in Plant Histidine Biosynthesis: Arabidopsis myoinositol monophosphatase-like2 Encodes a Functional Histidinol-Phosphate Phosphatase

Histidine (His) plays a critical role in plant growth and development, both as one of the standard amino acids in proteins, and as a metal-binding ligand. While genes encoding seven of the eight enzymes in the pathway of His biosynthesis have been characterized from a number of plant species, the identity of the enzyme catalyzing the dephosphorylation of histidinol-phosphate to histidinol has remained elusive. Recently, members of a novel family of histidinol-phosphate phosphatase proteins, displaying significant sequence similarity to known myoinositol monophosphatases (IMPs) have been identified from several Actinobacteria. Here we demonstrate that a member of the IMP family from Arabidopsis (Arabidopsis thaliana), myoinositol monophosphatase-like2 (IMPL2; encoded by At4g39120), has histidinol-phosphate phosphatase activity. Heterologous expression of IMPL2, but not the related IMPL1 protein, was sufficient to rescue the His auxotrophy of a Streptomyces coelicolor hisN mutant. Homozygous null impl2 Arabidopsis mutants displayed embryonic lethality, which could be rescued by supplying plants heterozygous for null impl2 alleles with His. In common with the previously characterized HISN genes from Arabidopsis, IMPL2 was expressed in all plant tissues and throughout development, and an IMPL2:green fluorescent protein fusion protein was targeted to the plastid, where His biosynthesis occurs in plants. Our data demonstrate that IMPL2 is the HISN7 gene product, and suggest a lack of genetic redundancy at this metabolic step in Arabidopsis, which is characteristic of the His biosynthetic pathway.His is one of the standard 20 amino acids found in proteins, and is required for plant growth and development (Muralla et al., 2007; Bikard et al., 2009). The occurrence of His in the active sites of numerous enzymes is attributable to the imidazole functional group (pK_a approximately 6), which can alternate between the protonated and unprotonated states under physiologically relevant conditions, allowing its participation in acid-base catalysis (Fersht, 1999). His also plays important biochemical roles as a nucleophile in phosphoryl group transfer, and as a metal-binding ligand (Fraústo da Silva and Williams, 2001; Harding, 2004).Genetic analysis of His biosynthesis has provided insights into key regulatory mechanisms in microorganisms, notably the discovery of operon structure and metabolic regulation through attenuation (Ames et al., 1960; Roth and Ames, 1966). However, this was the last amino acid biosynthetic pathway to be characterized in plants, and only recently has it been demonstrated that His biosynthesis follows the same route as that in microorganisms with 10 reactions catalyzed by eight enzymes (Fig. 1), beginning with the condensation of ATP and 5-phosphoribosyl-1-pyrophosphate catalyzed by the enzyme ATP-phosphoribosyl transferase (Ohta et al., 2000). His biosynthesis is linked to purine metabolism through its precursors 5-phosphoribosyl-1-pyrophosphate and ATP, and the release of an intermediate (5′-phosphoribosyl-4-carboxamide-5-aminoimidazole) at the branch point step catalyzed by imidazole-glycerol phosphate synthase, which enters the de novo purine synthesis pathway (Alifano et al., 1996; Ward and Ohta, 1999). While the regulation of His biosynthesis has not been as comprehensively investigated in plants as it has in microorganisms, analysis of transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing single HISN genes under the control of the cauliflower mosaic virus 35S promoter suggests that ATP-phosphoribosyl transferase activity controls the size of the pool of free His in plants (Ingle et al., 2005; Rees et al., 2009).Open in a separate windowFigure 1.The His biosynthetic pathway in plants. Abbreviations used for enzyme names in this work are shown in parentheses, and enzyme commission numbers and Arabidopsis gene loci indicated. Modified after Ward and Ohta (1999), Ingle et al. (2005), and Muralla et al. (2007).Genes encoding seven of the eight enzymes in the His pathway were identified in plants during the late 1990s (Nagai et al., 1993; Mori et al., 1995; El Malki et al., 1998; Fujimori and Ohta, 1998a, 1998b; Fujimori et al., 1998; Ohta et al., 2000) and, in contrast to most other amino acid biosynthetic pathways, the majority were found to be encoded by single genes in the Arabidopsis genome (Stepansky and Leustek, 2006). The missing link in the pathway has been HISN7, histidinol-phosphate phosphatase (HPP; EC 3.1.3.15). While dephosphorylation of histidinol-P to histidinol was first detected in plant extracts almost 40 years ago (Wiater et al., 1971), the identity of the enzyme(s) catalyzing this reaction in plants is unknown. HPP proteins identified from microorganisms prior to 2006 can be grouped into one of two superfamilies—the DDDD (which contain four invariant Asp residues) and PHP (polymerase and histidinol phosphatase) superfamilies (Lee et al., 2008). The DDDD superfamily contains bifunctional enzymes from enteric bacteria (such as Escherichia coli) where dephosphorylation of histidinol-P is catalyzed by the N-terminal domain, while the C terminus displays imidazole glycerol-phosphate dehydratase (IGPD) activity (Alifano et al., 1996; Fani et al., 2007), and monofuctional HPP proteins such as that recently identified in Thermococcus onnurineus (Lee et al., 2008). In contrast, all members of the PHP family identified to date are monofunctional HPP enzymes, including those encoded by HIS2 in Saccharomyces cerevisiae and ytvP in Bacillus subtilis (Alifano et al., 1996; le Coq et al., 1999). Notably, neither the Arabidopsis nor rice (Oryza sativa) genomes contain any sequences with significant sequence identity to members of either the DDDD or PHP superfamilies (Stepansky and Leustek, 2006).A novel HPP protein, showing no sequence similarity to members of either the DDDD or PHP superfamilies, was recently isolated from Corynebacterium glutamicum (Mormann et al., 2006), and orthologs have subsequently been identified in other Actinobacteria, including Streptomyces coelicolor (Marineo et al., 2008). All members of this new family display significant sequence similarity to known myoinositol monophosphatases (IMPs; Mormann et al., 2006), which catalyze the hydrolysis of the ester bond of d-myoinositol 1(or 3)-P (d-Ins 1-P, d-Ins 3-P) to generate myoinositol, without the production of a phospho-enzyme intermediate (Leech et al., 1993). In eukaryotes, myoinositol plays a crucial role in a number of cellular processes including phosphatidylinositol-mediated signaling and the membrane anchoring of proteins (Boss et al., 2006; Fujita and Jigami, 2008). To date, three putative IMP encoding sequences have been identified in the Arabidopsis genome: VTC4 (At3g02870), myoinositol monophosphatase-like1 (IMPL1; At1g31190), and IMPL2 (At4g39120; Torabinejad et al., 2009). VTC4 was first reported as the l-Gal 1-P phosphatase in ascorbate biosynthesis in Arabidopsis (Laing et al., 2004; Conklin et al., 2006), but was recently demonstrated to be a bifunctional enzyme also able to catalyze the dephosphorylation of d-Ins 1-P and d-Ins 3-P to myoinositol in vitro (Torabinejad et al., 2009). vtc4 null mutants showed only a 30% reduction in myoinositol content, suggesting genetic redundancy in the capacity to generate myoinositol, and accordingly IMPL1 and IMPL2 were shown to have in vitro IMP activity (Torabinejad et al., 2009). Both enzymes were also able to utilize l-Gal 1-P as a substrate, suggesting some promiscuity in their substrate specificity. Here we demonstrate that the IMPL2 protein encoded by At4g39120 is also able to catalyze the dephosphorylation of histidinol-P to histidinol, completing the His biosynthetic pathway in plants.     

Catechol estrogen quinones as initiators of breast and other human cancers: implications for biomarkers of susceptibility and cancer prevention

Exposure to estrogens is associated with increased risk of breast and other types of human cancer. Estrogens are converted to metabolites, particularly the catechol estrogen-3,4-quinones (CE-3,4-Q), that can react with DNA to form depurinating adducts. These adducts are released from DNA to generate apurinic sites. Error-prone base excision repair of this damage may lead to the mutations that can initiate breast, prostate and other types of cancer. The reaction of CE-3,4-Q with DNA forms the depurinating adducts 4-hydroxyestrone(estradiol) [4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua. These two adducts constitute more than 99% of the total DNA adducts formed. Increased levels of these quinones and their reaction with DNA occur when estrogen metabolism is unbalanced. Such an imbalance is the result of overexpression of estrogen activating enzymes and/or deficient expression of the deactivating (protective) enzymes. This unbalanced metabolism has been observed in breast biopsy tissue from women with breast cancer, compared to control women. Recently, the depurinating adduct 4-OHE1(E2)-1-N3Ade has been detected in the urine of prostate cancer patients, but not in urine from healthy men. Mutagenesis by CE-3,4-Q has been approached from two different perspectives: one is mutagenic activity in the lacI reporter gene in Fisher 344 rats and the other is study of the reporter Harvey-ras gene in mouse skin and rat mammary gland. A-->G and G-->A mutations have been observed in the mammary tissue of rats implanted with the CE-3,4-Q precursor, 4-OHE2. Mutations have also been observed in the Harvey-ras gene in mouse skin and rat mammary gland within 6-12 h after treatment with E2-3,4-Q, suggesting that these mutations arise by error-prone base excision repair of the apurinic sites generated by the depurinating adducts. Treatment of MCF-10F cells, which are estrogen receptor-alpha-negative immortalized human breast epithelial cells, with E2, 4-OHE2 or 2-OHE2 induces their neoplastic transformation in vitro, even in the presence of the antiestrogen ICI-182,780. This suggests that transformation is independent of the estrogen receptor. The transformed cells exhibit specific mutations in several genes. Poorly differentiated adenocarcinomas develop when aggressively transformed MCF-10F cells are selected and injected into severe combined immune depressed (SCID) mice. These results represent the first in vitro/in vivo model of estrogen-induced carcinogenesis in human breast epithelial cells. In other studies, the development of mammary tumors in estrogen receptor-alpha knockout mice expressing the Wnt-1 oncogene (ERKO/Wnt-1) provides direct evidence that estrogens may cause breast cancer through a genotoxic, non-estrogen receptor-alpha-mediated mechanism. In summary, this evidence strongly indicates that estrogens can become endogenous tumor initiators when CE-3,4-Q react with DNA to form specific depurinating adducts. Initiated cells may be promoted by a number of processes, including hormone receptor stimulated proliferation. These results lay the groundwork for assessing risk and preventing disease.