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Human Ads (adenoviruses) have been extensively utilized for the development of vectors for gene transfer, as they infect many cell types and do not integrate their genome into host-cell chromosomes. In addition, they have been widely studied as cytolytic viruses, termed oncolytic adenoviruses in cancer therapy. Ads are non-enveloped viruses with a linear double-stranded DNA genome of 30–38 kb which encodes 30–40 genes. At least 52 human Ad serotypes have been identified and classified into seven species, A–G. The Ad capsid has icosahedral symmetry and is composed of 252 capsomers, of which 240 are located on the facets of the capsid and consist of a trimeric hexon protein and the remaining 12 capsomers, the pentons, are at the vertices and comprise the penton base and projecting fibre protein. The entry of Ads into human cells is a two-step process. In the first step, the fibre protein mediates a primary interaction with the cell, effectively tethering the virus particle to the cell surface via a cellular attachment protein. The penton base then interacts with cell-surface integrins, leading to virus internalization. This interaction of the fibre protein with a number of cell-surface molecules appears to be important in determining the tropism of adenoviruses. Ads from all species, except species B and certain serotypes of species D, utilize CAR (coxsackie and adenovirus receptor) as their primary cellular-attachment protein, whereas most species B Ads use CD46, a complement regulatory protein. Such species-specific differences, as well as adaptations or modifications of Ads required for applications in gene therapy, form the major focus of the present review.
Over the last 50 years, significant progress has been made towards understanding the molecular biology of Ads (adenoviruses), and these studies have also provided major insights into fundamental aspects of DNA replication, RNA splicing, oncogenic transformation and apoptosis [1,2]. At present, at least 52 different human Ad serotypes have been identified which have been classified into seven species, A–G, mainly on the basis of haemagglutination properties and phylogenetic analysis (Table 1) [3].
|Species .
|Serotypes .
|Commonly associated disease .
|Primary attachment molecule .
|Oncogenic potential (in rodents) .
|GC content of genome (%) .
|Number of shaft repeats in fibre* .
|12, 18, 31
|Respiratory (B:1) or urinary tract disease (B:2)
|1, 2, 5, 6
|Respiratory disease
|8, 9, 10, 13, 15, 17, 19, 20, 22–30, 32, 33, 36–39, 42–49, 51
|CAR (sialic acid for 8, 19, 37 and CD46 for 37)
|Respiratory disease/conjunctivitis
|40, 41
|CAR (for long fibres; short fibres unknown)
|Species .
|Serotypes .
|Commonly associated disease .
|Primary attachment molecule .
|Oncogenic potential (in rodents) .
|GC content of genome (%) .
|Number of shaft repeats in fibre* .
|12, 18, 31
|Respiratory (B:1) or urinary tract disease (B:2)
|1, 2, 5, 6
|Respiratory disease
|8, 9, 10, 13, 15, 17, 19, 20, 22–30, 32, 33, 36–39, 42–49, 51
|CAR (sialic acid for 8, 19, 37 and CD46 for 37)
|Respiratory disease/conjunctivitis
|40, 41
|CAR (for long fibres; short fibres unknown)
Number of fibre shaft repeats found for individual serotypes within each species, not necessarily representative of all serotypes within a particular species.
Much of our current understanding of Ad interactions with the host has been based on studies using the species C Ads, namely serotypes 2 and 5 (Ad2 and Ad5). The well-characterized molecular biology of these viruses has made them a frequent choice for gene therapy vector development, as has their ability to transduce a broad range of dividing and non-dividing cells with high efficiency. In addition, the genome of Ads is relatively easy to manipulate and can carry a transgene of up to 30 kb. It also does not integrate into the human genome, minimizing the risk of insertional mutagenesis. Currently, a major application for Ad-based gene therapy is cancer treatment. Replication-deficient Ad vectors have been used to introduce tumour suppressor genes, inhibit oncogenes, modulate tumour-specific immune responses and to introduce 'suicide' genes into tumours to induce cytotoxic effects. However, studies using these vectors have shown limited clinical efficacy due to the restricted access of these vectors to the majority of cancer cells within the three-dimensional tumour mass [4]. A much more promising approach to cancer treatment harnesses the selective replication of viruses in tumour cells to facilitate virus-mediated cytolysis. Released progeny virus can then spread to and infect neighbouring cells, enabling the virus to spread and lyse cells throughout the entire tumour [5]. However, the species C Ads used in these studies have not yet shown clinical benefit due to limited efficiency of infection, therefore alternative species of Ads are being investigated for their oncolytic potential. The species B Ads are one such species which, when compared with Ad5 vectors, show improved virus entry into a wide range of cancer cells [6].
Although much of the structure and biology of species B Ads is similar to that of the species C viruses, there are aspects of the species B virus life cycle which differ and remain to be defined. With increasing interest in developing species B Ads as oncolytic agents, it is critical to obtain a detailed understanding of the interactions of these viruses with the host. The present review will provide an up-to-date overview of the molecular biology of species C Ads, highlight known features which differ in species B virus infection and assess the impact of these differences on the use of these viruses as gene therapy and oncolytic agents.
Ads are non-enveloped viruses of approx. 90 nm in diameter, with a linear, non-segmented, double-stranded DNA genome of 30–38 kb. At the ends of each Ad DNA molecule are ITRs (inverted terminal repeats) which range from 36 bp to more than 200 bp, depending on the serotype, and are important during genome replication. Each 5′ terminus of the virus DNA is covalently linked to a TP (terminal protein) which plays a key role in the initiation of virus DNA replication [5]. The Ad genome, while showing marked sequence diversity between the different species, is organized in a distinct manner which is highly conserved [7]. It is usually divided into regions which encode the early (E) genes, E1A, E1B, E2A, E2B, E3 and E4, the delayed early genes, IX and IVa2, and late (L) genes, L1–L5. In addition, other minor transcription units are present in the viral genome [2]. The genome map comprises 100 map units, with the E1A gene conventionally placed at the left end of the genome (Figure 1). There are also two genes encoding virus-associated RNAs I and II (VAI and VAII RNAs) which function in the inhibition of host protein synthesis in infected cells. Expression of Ad genes is temporally regulated, with early genes being expressed soon after the virus enters cells, before the onset of viral DNA replication. Proteins encoded by these genes are mainly involved in the regulation of viral DNA replication and transcription. Late genes are usually active after replication of viral DNA and encode the structural proteins of the virus particle.
Ad genome organization
The icosahedral Ad particle is composed of 13 structural proteins, some of which are denoted by roman numerals (II–X), and comprises 252 capsomeres of which 240 consist of trimeric hexon protein (polypeptide II) and the remaining 12 capsomeres are pentons [5]. Pentons, each of which consists of a pentamer of penton base (polypeptide III) from which projects a trimeric fibre protein (polypeptide IV), are found at each of the 12 vertices. The penton bases are important for the stabilization of the capsid. The minor components of the outer capsid shell (polypeptides IIIa, VI, VIII and IX) have been located by cryo-EM (cryo-electron microscopy); their structures and functions have been reviewed previously [5,8]. Of particular note is the external location of polypeptide IX, which provides an opportunity for genetic modification resulting in altered virus vector tropism [3,8]. Inside the capsid, the virus DNA is closely associated with basic proteins, polypeptides V, VII and X (also termed Mu), forming the virus core. Polypeptide IVa2 is an ATP-binding protein and binds specific virus DNA sequences near the left terminus, mediating genome packaging into capsid precursors [5]. The capsid also contains a virus-encoded protease (adenain) which cleaves precursors to several capsid proteins (IIIa, VI, VII, VIII, X and TP) and is necessary for assembly of infectious virus particles [5]. The overall structure of the capsid, indicating the location of the major and minor capsid proteins, is shown schematically in Figure 2 and the role of each capsid protein is summarized in Table 2.
Structural proteins associated with the Ad capsid
|Polypeptide/name .
|Location .
|Function .
|II (hexon)
|Facets of icosahedron
|Major structural component, forms facets of the capsid
|III (penton base)
|Capsid vertices
|Contains an RGD motif which facilitates interaction with cellular integrins*
|Underside of penton base
|Stabilizes the vertices
|IV (Fibre)
|Projecting from the penton base
|Mediates the initial attachment to host cells
|Links core to capsid, possibly aids nuclear localization
|Inner hexon cavity
|Protease cofactor, assembly, endosome disruption and nuclear import of hexon
|Targets viral genome to the nucleus and condenses DNA
|Between hexons
|Stabilization of peripentonal hexon–hexon interactions
|External faces of the capsid
|Stabilization of virion. Transcriptional activator
|5′-End of the genome
|Primes DNA replication
|DNA condensation
|DNA packaging
|Ad protease
|Cleaves precursor proteins
|Polypeptide/name .
|Location .
|Function .
|II (hexon)
|Facets of icosahedron
|Major structural component, forms facets of the capsid
|III (penton base)
|Capsid vertices
|Contains an RGD motif which facilitates interaction with cellular integrins*
|Underside of penton base
|Stabilizes the vertices
|IV (Fibre)
|Projecting from the penton base
|Mediates the initial attachment to host cells
|Links core to capsid, possibly aids nuclear localization
|Inner hexon cavity
|Protease cofactor, assembly, endosome disruption and nuclear import of hexon
|Targets viral genome to the nucleus and condenses DNA
|Between hexons
|Stabilization of peripentonal hexon–hexon interactions
|External faces of the capsid
|Stabilization of virion. Transcriptional activator
|5′-End of the genome
|Primes DNA replication
|DNA condensation
|DNA packaging
|Ad protease
|Cleaves precursor proteins
Exceptions include the species F Ads
Together, the fibre and the penton base proteins mediate uptake of the virus into host cells; the fibre by initial attachment to the cell and penton base by subsequent interaction with cell-surface integrins. The penton base from species A, B, C, D, E and G contains an RGD (arginine-glycine-aspartate) motif within one of the exterior loops in the upper part of the penton base which facilitates interaction with integrins, leading to uptake of the virus into cells [9].
The capsids of most serotypes contain only one type of fibre; however, species F serotypes carry a mixture of both short and long fibres [10,11]. The species G serotype, Ad52, also encodes two fibre proteins [12]. All fibre proteins consist of three distinct regions: an N-terminal tail domain, a shaft region of variable length and a C-terminal knob (or head) domain. Structural conservation between fibres of different serotypes is high, despite a considerable degree of sequence variation. The N-terminus of the fibre contains a highly conserved sequence (FNPVYPY) which interacts with the penton base by several hydrogen bonds and a salt bridge [13,14]. Although there are five potential fibre-binding sites in the pentameric penton base, only three fibre polypeptide chains can attach, due to steric hindrances [9]. The fibre adopts a trimeric structure, which requires sequences from both the fibre shaft and the knob domain [15]. The fibre shaft contains a number of repeating units of 15–20 amino acids, each of which is 13.2 Å (1 Å=0.1 nm) in length and contains β-sheet structures [16,17]. The structural motif within the shaft has been defined as a triple β-spiral in which the repeats from each fibre monomer intertwine to form a trimeric shaft region which is stable yet flexible [18]. The lengths of fibre shafts vary between serotypes (Table 1), ranging from very short fibre shafts consisting of only six repeating units (such as that found for Ad35 and other species B viruses) to long fibres which carry 22 (Ad5) or 23 repeating units (Ad12) [19]. The length, as well as the flexibility of this region, is important in mediating interactions with the cell surface [20]. The globular knob domain is found at the distal end of the fibre shaft (Figure 3). The crystal structures of fibre knobs from several serotypes have been solved, including Ad2, Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21 and Ad35 [21–28]. The knob from each monomer contains eight-stranded anti-parallel β-barrels which, following trimerization, interact to form a characteristic 'three-bladed propeller' structure [18,22,23]. The trimeric knob contains a depression centred along the 3-fold axis with valleys found between each of the subunits. The loops which project from each fibre knob monomer play important roles in interactions with cell-surface molecules.
Three-dimensional structure of the Ad fibre protein
Ad infection involves several steps before the virus is delivered to the nucleus, which is initiated by binding to the cell surface and entry into the cell. The virus then escapes from the endosome and translocates to the nucleus (Figure 4). The pathway described for the species C Ads (i.e. Ad2 and Ad5) has been studied in great depth and has been termed the 'classical' pathway of infection [29]. However, major differences have been identified between the entry of the species B viruses compared with the 'classical' pathway used by species C viruses, principally due to the recognition of alternative cell-surface attachment molecules. It should also be noted that, in addition to the differences in intracellular trafficking observed between different Ad species, factors such as cell type and cell-cycle stage can also influence the entry pathway followed [30].
The entry pathway of species C Ads
Molecules which mediate Ad attachment to the cell surface
Knowledge of the interaction of Ads with sites on the cell surface has increased dramatically in recent years, largely as a result of the determination of the structures of capsid proteins by X-ray crystallography. Several cell-surface attachment molecules for Ads have been identified and the precise amino acids which mediate interactions with virus proteins have been defined. Although receptor-independent entry exists for certain serotypes, interaction with a primary cellular attachment molecule is likely to form the major route for Ad entry and is mediated by the C-terminal knob domain of the fibre protein.
CAR (coxsackie and Ad receptor): a major Ad attachment molecule
Early studies showed that Coxsackie virus B3 (an RNA virus) and Ad2 competed for the same attachment site on human cells [31]. A monoclonal antibody that blocked Coxsackie B virus attachment to cells was used to isolate a 46 kDa protein which was subsequently shown to serve as an attachment molecule for Coxsackie B viruses, as well as Ad2 and Ad5 [32]. The 46 kDa protein has been termed CAR and is generally found located at, or close to, tight junctions in polarized epithelial cells where it functions as a cellular-adhesion molecule [33]. CAR mRNA is produced by RNA splicing, generating a major mRNA comprising seven exons (termed CAR Ex7). Levels of this mRNA show considerable variation in human tissues, with expression detected in the liver, kidneys, lungs, brain, heart, colon, small intestine, prostate, testes and pancreas [34]. Homologues of CAR are found in a number of species including the cow, pig, dog, rat and mouse [35]. The receptor contains two extracellular immunoglobulin-like domains: CAR-D1 and CAR-D2. In addition, CAR contains a single membrane-spanning region which connects the extracellular domains to the C-terminal cytoplasmic domain [32].
Serotypes from most of the Ad species have been shown to utilize CAR, with the exception of the species B viruses [36]. The crystal structure of the Ad12 fibre knob in complex with CAR-D1 demonstrated that four loop structures from the fibre mediate the interaction with CAR-D1, with the AB-loop contributing to more than 50% of the interaction. Three hydrogen bonds formed between the AB-loop and CAR in particular add to the stability of the interaction [37]. The DE loop is also involved in the interaction [37,38]. Salt bridges, hydrogen bonds and Van der Waals forces all contribute to these interactions.
Although CAR has been shown to support Ad entry to cultured cell lines, in the airway epithelium of the host, CAR expression is restricted to tight junctions and the basolateral membrane. Therefore there has been some debate as to whether CAR is accessible to the virus in order to facilitate attachment in the human host. It has also been proposed that the main function of the CAR–fibre interaction may be to support viral escape, rather than to promote attachment. As CAR forms homophilic interactions at tight junctions, excess fibre proteins produced during the infectious life cycle may disrupt CAR–CAR homodimers, thereby promoting release of progeny virus to the airway lumen [39]. A recent report has identified low levels of a novel CAR splice variant containing an additional eighth exon (CAR Ex8), encoding a protein product with an additional 13 amino acids at the C-terminus which has been detected on the apical surface of polarized airway epithelial cells and may be responsible for mediating the initial interactions with the virus [40].
Additional cell-surface molecules have also been proposed to function as attachment sites for certain species C Ads including VCAM-1 (vascular cellular adhesion molecule-1) and HS-GAGs (heparan sulfate glycosaminoglycans). However, the extent to which these interactions are involved during infection remains to be determined [41,42].
Role of blood factors in Ad tropism
It has become increasingly evident that Ad5 biodistribution in animals does not directly correlate with the expression of CAR, with significant levels of liver transduction detected in mouse models following systemic delivery of the virus [43–45]. Intravenous delivery of Ads has been utilized in gene therapy studies, although dissemination of Ad to the bloodstream during normal infection is rare. Unless the target of the gene therapy is the liver, high levels of liver transduction are generally undesirable as it not only directs the vector away from target organs but also, following administration of high levels of vector, can result in liver toxicity [46]. Several studies demonstrated that mutation of the fibre, resulting in ablation of CAR binding, had no effect on the resulting biodistribution or hepatotoxicity which followed systemic delivery of Ad5 to mice, suggesting that liver transduction occurred independently of CAR [47,48].
It was later demonstrated using binding studies and MS that the blood coagulation Factor IX and complement component C4BP (C4-binding protein) could interact with Ad5 [49]. Binding of these factors to the surface of Ad5 led to a proposed uptake mechanism in which these factors 'bridged' Ad5 to HSPGs (heparan sulfate proteoglycans) and low-density lipoprotein receptor-related proteins on the surface of hepatocytes. A combination of in vivo and in vitro studies identified additional blood factors [FX (Factor X), protein C and Factor VII] which could also enhance the transduction of hepatocytes by Ad5 [50]. In particular, treatment of mice with warfarin, a drug that prevents the maturation and secretion of vitamin K-dependent coagulation factors (including FX), significantly reduced liver toxicity. This study also demonstrated, using SPR (surface plasmon resonance), that FX (which had the most significant effect on Ad5 uptake) bound to the capsid surface [50]. FX is a zymogen of a vitamin K-dependent serine protease which contains one Gla (γ-carboxyglutamic) domain, two EGF (epidermal growth factor)-like domains and a serine protease domain. FX was also found to bind to and enhance the transduction of liver cells by Ad5 vectors containing species D fibre proteins, providing further evidence of the general importance of this coagulation factor in Ad liver toxicity [51].
Initially it was proposed that coagulation factor-mediated entry was likely to be mediated by fibre proteins, as reduced liver transduction was found with Ad5 vectors expressing short shaft-containing fibres [49,52,53]. However, SPR studies indicated that 'fibreless' Ad5 particles interacted as efficiently with FX as wild-type Ad5, questioning the involvement of fibre protein [54]. The same study also demonstrated that the major binding site for FX on the Ad5 capsid was the hexon and that this interaction required calcium. Cryo-EM analysis revealed that FX binds within the central depression of the hexon trimer. In addition, SPR analysis demonstrated that certain Ad serotypes interacted with FX with high affinity (Ad2, Ad4, Ad16 and Ad50), whereas others displayed weak affinity (Ad3 and Ad35) and some formed no interaction (Ad26 and Ad48) [54]. A similar study confirmed that the interaction between Ad5 hexon and FX occurred with picomolar affinity [55]. Finally, specific residues have been identified in the HVRs (hypervariable regions) of Ad5 hexon, that contribute to its high affinity for FX [56].
The studies on ablation of the CAR-binding site in the fibre and coagulation factor binding (described above) appear to suggest that CAR plays a minor role in whole organisms (principally rats and mice) in mediating entry of species C viruses when administered intravenously, and that the entry process is mainly determined by interaction with soluble factors such as FX. It is worth adding that when human CAR was ubiquitously expressed in a transgenic mouse, the biodistribution of Ad5 vectors was greatly increased, and lymphoid, myeloid and endothelial cells were targeted, as well as the brain and lungs [57]. This may suggest that there are differences in interaction of human Ads with rodent, compared with human CAR and that rodents may not necessarily recapitulate all the features of human Ad entry.
CD46: an attachment molecule for the species B Ads
Experiments involving binding of soluble CAR to purified virus particles and cross-blocking of virus entry with recombinant fibre proteins showed that the species B viruses do not use CAR for entry into human cells [36]. The species B Ads have been classified into two sub-species: the B:1 viruses (serotypes 3, 7, 16, 21 and 50) and the B:2 viruses (11, 14, 34 and 35) [58]. This division was made on the basis of conservation of restriction endonuclease-cleavage sites, but also broadly correlated with the cell tropism of these viruses: the B:1 viruses causing infection of the upper respiratory tract, whereas the B:2 viruses are associated with infection of the kidneys and urinary tract [59]. This also indicated that these two sub-species may utilize alternative cell-surface attachment sites [59]. This possibility was investigated using cross-competition studies with radiolabelled viruses from serotypes representing the B:1 sub-species (Ad3 and 7) and the B:2 viruses (Ad11 and 35) [60]. Interestingly, whereas Ad11 efficiently blocked the interactions of serotypes from both sub-species with human cells, Ad3 only completely blocked the interaction of Ad3 and Ad7 viruses and partially inhibited the binding of the B:2 viruses. This suggested the existence of two potential species B attachment molecules, one of which could be utilized by all viruses of species B and another that was specifically used by the B:2 viruses. The molecule(s) common to all species B viruses was termed sBAR (species B Ad receptor), whereas the B:2-specific molecule(s) was denoted sB2AR (species B2 Ad receptor) [60].
Characterization of cell-surface molecules that interacted with species B viruses revealed CD46 as a major attachment molecule for certain serotypes. Several different, but complementary, approaches were utilized in those investigations. Affinity purification and mass spectrometric analysis of proteins from lysates of human cells that bound to recombinant Ad35 fibre protein showed CD46 to be a major binding partner of Ad35 and several other species B viruses, with the possible exception of Ad3 and Ad7 [61]. In another study, a cDNA expression library from human K562 leukaemia cells was introduced into a hamster cell line which is non-permissive for species B Ad infection. This generated clones of cells that were susceptible to Ad3 infection, most of which expressed CD46, suggesting that this molecule was required for Ad3 uptake [62]. Finally, taking advantage of the fact that Ads and picornaviruses interact with cell-surface Ig-domain proteins, a screen of previously described proteins that mediate picornavirus entry (specifically CD46 and CD55) was performed and identified a requirement for CD46 in Ad11 entry [63]. While all three experimental approaches identified CD46 as a major protein involved in species B:2 virus entry, there was a lack of consensus regarding the role of CD46 in sub-species B:1 (Ad3 and Ad7) entry; indeed, a subsequent study that addressed this point identified a role for CD46 in mediating Ad of all of the species B viruses, with the important exception of Ad3 and Ad7 [64].
CD46 has been described as a 'pathogen magnet' since it is utilized by a number of viral (measles virus and human herpesvirus-6) and bacterial pathogens (Neisseria and Streptococcus species) to enter human cells [65]. CD46 plays a role as an inhibitory complement receptor, preventing the destruction of host tissues by binding C3b and C4b and acting as a cofactor for their Factor I-mediated degradation, and is expressed by almost all human nucleated cells [66]. Four major isoforms of this protein have been described. The extracellular N-terminal region of each isoform is identical, containing four SCRs (short consensus repeats). This is followed by a region rich in serine, threonine and proline residues and is referred to as the STP domain. The STP domain is connected to one of two possible cytoplasmic domains by a hydrophobic transmembrane domain. The four main CD46 isoforms are termed C1, C2, BC1 and BC2 which represent the different regions within the STP domains (either C alone or B and C) in conjunction with either the type 1 or type 2 cytoplasmic domains. The relative ratio of these different isoforms on the cell surface can vary between individuals, with 65% of the population expressing predominantly BC types, 30% displaying equal amounts and 5% showing greater expression of the C types [67]. CD46 is constitutively internalized by clathrin-coated pits and recycled to the cell surface, with a half-life of approx. 8–12 h. Cross-linking of CD46 at the cell surface by either ligand or antibody results in internalization by macropinocytosis [68].
Mutation of residues within CD46 indicated that N-terminal extracellular domains, namely the SCR domains, were responsible for mediating Ad35 contacts with CD46 [69–71]. A later study confirmed that another B:2 Ad, Ad11, also bound to CD46 via the SCR domains. In contrast with previous findings, species B:1 serotypes 3 and 7 were also reported to interact with the SCR1 and SCR2 regions of CD46, using expression of mutated CD46 SCR–CD4 transmembrane proteins [72]. This suggested that the B:1 and B:2 Ads interact with CD46 in a similar manner. Another group has shown that the B:2 serotypes interact with CD46 with much higher affinity than the B:1 viruses [27]; for example, comparisons made between Ad16, a species B:1 virus, and the B:2 virus Ad11 revealed that Ad16 had a 70-fold lower affinity for CD46 than Ad11 [27]. This difference was proposed to result from the presence of two additional residues within the FG loop of Ad16 fibre, a feature which is shared with Ad3 and Ad7. The replacement of Arg279 with a glutamine residue in the fibre knob domains of serotypes 7 and 14 has also been proposed to contribute to the reduced affinity of these serotypes for CD46 when compared with Ad11 [25]. Previous studies from this laboratory have shown that the expression of human CD46 on the surface of non-permissive CHO (Chinese-hamster ovary) cells supported the attachment and entry of Ad3 [73]. However, in order to achieve Ad3 entry, relatively high levels of CD46 expression were required. The expression of cell-surface CD46 at relatively low levels (comparable with those found on normal human cells) was unable to mediate Ad3 entry, suggesting a low affinity of Ad3 for CD46. In addition, evidence was obtained to indicate the presence of an additional, as yet unidentified, cell-surface molecule(s) in human epithelial cell lines which may be used in preference to CD46 [73]. Overall, while CD46 is a major attachment molecule for species B Ads, the affinity of the virus for binding to CD46 may determine whether CD46 is utilized, and increased levels of CD46 may overcome low-affinity interactions, as observed for Ad3 and Ad16. Finally, additional attachment molecules may remain to be discovered for species B viruses such as Ad3.
CD80 and CD86: alternative species B virus attachment molecules?
Using affinity chromatography, an 80 kDa protein was purified from HeLa cell lysates with Ad3F (Ad3 fibre) knob conjugated to Sepharose beads [74]. Analysis by MS and N-terminal peptide sequencing showed it to be CD86 (also known as B7.2). This protein displays high levels of sequence similarity to CD80 (B7.1) and it was therefore assumed that both CD80 and CD86 may interact with Ad3. Further studies suggested that all of the species B serotypes could interact with CD80 and CD86 [75]. CD80 and CD86 are co-stimulatory molecules which provide additional signals required for activation of T-cells and are present on antigen-presenting cells, such as macrophages, B-cells and dendritic cells. These cell types have not, hitherto, been identified as target cells for species B viruses [59]. Interestingly, transduction of malignant glioma cells by an Ad5 vector containing Ad3 fibres (Ad5/3F) was reported to involve the receptors CD80 and CD86, since antibodies against these molecules inhibited entry of Ad5/3 [76]. In contrast with these findings, transduction of CHO cells expressing high levels of CD80 or CD86 with an Ad3 virus carrying an EGFP (enhanced green fluorescent protein) transgene failed to provide evidence for the involvement of either co-stimulatory molecule in Ad3 entry, suggesting that other cell-surface molecules are required for Ad3 entry [73].
Cellular uptake of species C Ads
In the classical model, based on studies in cell culture, following the initial interaction between the knob domain of species C fibre proteins and the primary cellular receptor CAR, a second, lower-affinity, interaction between an RGD motif in the penton base and a cellular integrin has been proposed to occur [77–80]. Integrins are relatively large cell-surface molecules which are important for cellular attachment to the extracellular matrix and in cell signalling, involving aspects of cell migration, differentiation and growth. There are currently over 20 different members of the integrin family, each of which is a heterodimer consisting of α and β subunits [81]. Many integrins recognize an RGD motif in extracellular matrix proteins such as vitronectin and fibronectin. Integrins have been shown to mediate the attachment of a number of pathogens to cells including echovirus-1, foot-and-mouth-disease virus and cytomegalovirus, as well as bacteria belonging to the genus Yersinia [82–86]. An RGD motif is conserved in the protruding loops of penton base structures from all Ad serotypes, except those belonging to species F, and it has been demonstrated that Ads from species containing an RGD domain can interact with integrins [79,87]. The penton base interaction may occur between one of a number of RGD-binding integrins, but primarily with the αvβ3 and αvβ5 integrins [80].
As each penton base exists as part of a pentameric complex, each penton capsomere is capable of binding five integrin molecules. This property promotes integrin clustering which triggers the signalling events required for internalization of the virus. During species C infection, interaction of the penton base with integrins leads to a rearrangement of the actin cytoskeleton and uptake of the virus via clathrin-coated vesicles. Initially, binding to integrins induces a conformational change in the integrin which activates p85/p110 phosphoinositide 3-kinase. This stimulates Rac and CDC42 activation and the polymerization of actin monomers, which facilitates virus entry [88,89]. Entry via clathrin-coated vesicles was demonstrated by the inability of species C Ads to enter cells that carry a dynamin mutation [90,91]. Interestingly, fibre proteins from species C Ads have been proposed to be released from the capsid surface in a pH-independent manner following virus interactions with integrins [92–94].
Macropinocytosis: a species B Ad entry pathway
In contrast with species C Ads, entry of the species B:1 virus Ad3 to a range of cell types expressing a dynamin mutant was minimally affected, suggesting a low level of dynamin-dependent endocytosis in Ad3 infection. Ad3 has also been shown to stimulate membrane ruffling and fluid phase uptake through combined interaction with CD46 and integrins [95]. In addition, inhibitors of macropinocytosis prevented Ad3 entry [95]. Uptake of Ad3 therefore appears to occur primarily via macropinocytosis (Figure 5), although a very small proportion of virus (~1%) may be internalized by clathrin-mediated endocytosis. While macropinocytosis is an endocytic mechanism often associated with the non-selective uptake of fluid and membrane, a wide range of viruses have been reported to utilize this entry process including vaccinia virus, echovirus-1, Coxsackie B virus, Nipah virus and herpes simplex virus-1 [96,97]. Ad35, a species B:2 Ad, has also been shown to use macropinocytosis for entry into epithelial cells, suggesting a possible conserved entry pathway for the species B Ads [98]. Use of a range of specific inhibitors suggested that both Ad3 and Ad35 entry requires the sodium–proton exchanger, actin, PAK1 (p21-activated kinase 1), Rac, PKC (protein kinase C) and the transcriptional co-repressor CtBP1 (C-terminal binding protein 1 of E1A) (Figure 5). Similarly, both Ad3 and Ad35 co-localized with CD46 and αv integrins within macropinosomes [95,98]
The uptake of species B Ads into epithelial cells by macropinocytosis
Escape from the endosome
Following the internalization of species C Ads, the lumen of the endosome is rapidly acidified due to the activity of H+-vacuolar ATPase. This reduced pH has been proposed to induce conformational changes in the structure of Ad capsids, leading to the sequential release of capsid components [92]. Once any remaining fibre proteins have detached from the capsid surface, the additional vertex components, penton base and polypeptide IIIa, are released. This is followed by proteins VI and VIII, and finally the release of polypeptide IX [92]. The release of vertex proteins may be critical for endosomal escape, as demonstrated by a temperature-sensitive Ad2 mutant (ts1) which failed to release fibre proteins [99]. The N-terminal α-helix of polypeptide VI has been shown to be directly responsible for endosomal lysis. In mature virus particles, liberation of protein VI through the low-pH-mediated capsid disassembly triggers lysis of the endosome, allowing the disassembled structure to escape to the cytosol [100].
For species C viruses, escape from the endosome takes approx. 15 min. Although the process of disassembly for species B viruses is unclear, the escape of Ad3 from macropinosomes has been shown to be much slower than the endosomal release of species C viruses, taking approx. 30–40 min [95]. Ad5 viruses modified to carry species B Ad35 or Ad7 fibre proteins have also been shown to remain associated with late endosomes for up to 2 h after infection [101–103]. The tendency for species B capsids to accumulate in late endosomes, in contrast with the rapid translocation of species C viruses to the nucleus, may reflect a lower pH requirement by the species B viruses for disassembly [102,103].
Once released from the endosome, partially disassembled capsids must then reach the nucleus. For species C Ads, this translocation has been shown to involve microtubules [104–107]. Net movement towards the minus end of microtubules at speeds of approx. 1 μm/min is thought to depend on integrin signalling and the subsequent activation of p38 MAPK (mitogen-activated protein kinase) [108,109]. Cytoplasmic dynein has been shown to generate this movement towards the MTOC (microtubule-organizing centre), which for most cell types is located next to the nucleus [110]. Protein VI has also been implicated in the trafficking of disassembled capsids towards the nucleus. Protein VI contains a PPXY motif which is exposed during the early stages of capsid disassembly and recruits Nedd4 E3 ubiquitin ligases, resulting in ubiquitination of protein VI and rapid microtubule-dependent trafficking towards the nucleus [111]. Following arrival at the MTOC, the virus must detach from microtubules and associate with the NPC (nuclear pore complex). This is proposed to be mediated by the nuclear export factor CRM1 (chromosome region maintenance 1) or a nuclear factor exported by CRM1 [112].
Nuclear import
Ad docking with the NPC is mediated by hexon interactions with the NPC cytoplasmic filament protein CAN/Nup214 [113]. Ad particles are too large to diffuse through the NPC and therefore undergo further disassembly to release the viral DNA into the nucleus [114]. Following interaction with CAN/Nup214, soluble histone H1 binds to an acidic stretch of amino acids contained within one of the hexon surface loops. The histone H1 Imp7/Impβ import factor then binds to the H1 proteins which are bound to the Ad capsid. In addition, the molecular chaperone protein Hsc70 (heat-shock cognate 70 stress protein) has also been found to interact with the capsid to enhance infection [115]. Conformational changes which take place in the subvirion structure following interactions with histone H1 and nuclear-import factors enable protein VII to interact with transportin. This leads to the efficient nuclear import of proximal hexon–H1 complexes and the associated DNA and protein VII [113,116]. Inside the nucleus, TAF-1 (template-activating factor-1) binds to protein VII, remodelling virus chromatin to allow transcription of the genome [117]. While the hexon sequence engaged by histone H1 is conserved in all species C viruses, species B Ads do not carry a similar H1-interacting sequence in their hexon sequences and therefore may not share this import mechanism [113].
Assembly and release
Proteins produced from the translation of viral mRNA transcripts must be transported from the cytoplasm into the nucleus for particle assembly. Hexon monomers are assembled into trimers using the L4-100K protein as a scaffold [118]. Penton base pentamers and fibre trimers assemble independently in the cytoplasm and both structures contain an NLS (nuclear localization sequence) to direct nuclear import. The formation of penton capsomeres consisting of both the fibre and penton base is spontaneous and likely to occur in the nucleus [119]. Hexon trimers require association with protein VI for nuclear localization [120]. Inside the nucleus, L4-33K acts as a scaffolding protein to assist the assembly of capsids [121]. Localization of Ad3 and Ad5 fibre proteins in HeLa cells at 48 h post-infection reveals clear differences in the arrangement of these proteins between the two serotypes (Figure 6). In Ad5-infected cells, aggregates of pentons, consisting of both fibre and penton base proteins, have been suggested to represent privileged assembly platforms for the virus, aiding efficient assembly [122]. In contrast with the distinct fibre structures which are found in Ad5-infected cells, Ad3 proteins form much smaller aggregates which may suggest key differences in the mechanism of capsid assembly (Figure 6).
Intracellular location of Ad fibre in Ad3- and Ad5-infected HeLa cells
Following the formation of a procapsid, viral DNA is directed into the capsid at a unique vertex, in a polarized fashion. A packaging sequence which consists of seven AT-rich sequences found at the left end of the genome is thought to mediate the uptake of the Ad genome into the empty capsid [123]. This method of DNA packaging is analogous to that found for dsDNA (double-stranded DNA) bacteriophages such as PRD1, which uses an ATP motor to drive the incorporation of DNA into pre-formed capsids [124]. In Ad packaging, this activity may be provided by IVa2 [125]. After incorporation of viral DNA, the capsid can then be sealed prior to final maturation. IVa2, L1-52/55K and L4-22K have all been shown to bind to the packaging sequence, promoting this encapsidation process [126,127]. Final maturation of the assembled virus is performed by the Ad protease. The L3-23K cysteine protease (adenain) requires both DNA and the C-terminal fragment of protein VI for activity [128,129]. Once activated, the protease cleaves the precursors of polypeptides VI, VII, VIII, Mu and TP to yield an infectious particle. Although this mode of encapsidation is favoured, it should be noted that some studies have demonstrated that DNA packaging may be intimately linked with capsid assembly, suggesting that assembly may occur around a DNA-containing core [130].
Ad infection leads to lysis of infected cells which, following infection by species C Ads, results from the accumulation of the E3-11.6K protein, also termed the ADP (Ad death protein) [131]. ADP accumulates in cells during the late stages of infection and localizes to the nuclear membrane, ER (endoplasmic reticulum) and Golgi apparatus. In addition, the L3 protease cleaves cellular cytokeratin which disrupts cell integrity, making cells more susceptible to lysis [132]. The interaction of ADP with MAD2B protein is then proposed to bring about cell lysis, although this process is poorly understood [133]. It has also been proposed that Ads induce autophagy to facilitate the release of progeny virions rather than by cell lysis [134]. Interestingly, the species B Ads do not encode an ADP and therefore the mode of release of these viruses from infected cells remains to be determined.
Currently, Ads account for approx. 24% of all vectors used in gene therapy clinical trials. Early Ad vectors incorporated transgenes into the E1 region of the genome, rendering them replication-deficient. However, such vectors induced potent immune responses upon systemic application [135,136]. Responses are directed against both the vector capsid and the low levels of Ad capsid proteins expressed from the vector. Induction of immune responses limits transgene expression and reduces the capacity for vector re-administration. While the immunogenicity of these vectors may be reduced by the deletion of additional genes, the immunogenic nature of Ad vectors means that these viruses are better suited to applications for which prolonged transgene expression is not required. The use of Ad vectors in vaccination to protect against a range of infectious pathogens has therefore proved to be a common strategy [137]. A significant proportion of gene therapy studies have also been designed to target cancers, where only short-term gene expression is required.
The treatment of cancer cells using replication-deficient Ads may involve one of a number of strategies including the inactivation of oncogenes and the addition of tumour suppressor genes or apoptosis-inducing genes. However, these approaches have limited efficacy as they require the successful transduction of each cell within a tumour, which is currently not feasible. Another approach has been to construct replication-deficient Ads with genes encoding secreted factors such as GM-CSF (granulocyte macrophage colony-stimulating factor) and IL-12 (interleukin-12) and introduce them into tumours in order to modulate immune responses and stimulate cytotoxic effects towards the tumour [138]. Activation of apoptosis in non-transduced cells by soluble TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand) encoded by an Ad5 vector has also been reported [139]. As these responses occur in the vicinity of the transduced tumour cells, rather than at a specific cell, non-transduced cells are also susceptible [138,139]. Introduction of genes which encode prodrug-activating enzymes are also beneficial and lead to site-specific activation of drugs in the area of the tumour [140]. Although such studies have shown moderate success, recent developments have been directed away from these replication-deficient vectors and have focused upon the development of replicating Ads with oncolytic potential.
Oncolytic viruses
'Oncolytic virotherapy' describes an approach to cancer treatment which uses the selective replication of a virus to facilitate the lysis of cancer cells while sparing normal cells. Following lysis of infected tumour cells, progeny virions are released which can subsequently infect neighbouring tumour cells, allowing the spread of the oncolytic virus throughout the entire tumour. The cancer-killing property of viruses was recognized many years ago, but it was only recently that significant progress was made in the development of replication-competent viral vectors for clinical benefit [141]. When using a virus for oncolytic therapy, it is important that the virus transduces cancer cells efficiently and replicates specifically in those cells to promote efficient tumour cell lysis [142,143]. However, it is equally important to minimize damage to healthy tissues.
Ads replicate more efficiently in tumour cells compared with normal cells, which may be due to the increased metabolic activity of tumour cells [144]. However, specific replication of Ad vectors in cancer cells compared with normal tissues can be promoted by the mutation or removal of the viral E1A or E1B oncogenes. These Ad oncogenes encode proteins that modulate host cell signalling pathways through interaction with the protein products of the Rb (retinoblastoma) and p53 tumour suppressor genes respectively, to promote cell-cycle progression which favours virus replication. In tumour cells, cell growth is unregulated, with many human cancers harbouring mutations in p53 or Rb. Ad vectors that lack E1B-55K or contain mutations in E1A are unable to inactivate, respectively, the p53 and Rb proteins found in normal cells and therefore display restricted replication in normal cells. However, they can replicate efficiently in cells in which p53 or Rb is mutated, i.e. tumour cells [145]. Modification of Ads in this manner therefore confers conditional replication which is limited to tumours. Replication-competent Ad can be further modified to ensure that replication only occurs within tumour cells by the inclusion of tumour-specific promoter elements in the vector [146]. The promoter for the PSA (prostate-specific antigen) is one such example which can be used to replace the Ad E1A promoter, thereby restricting Ad vector replication to PSA-expressing prostate cancer cells [147].
Oncolytic viruses have shown considerable potential in cancer biotherapy, either in combination with transgenes or combined with conventional treatments such as chemotherapy and radiotherapy [148–150]. However, oncolytic Ads, which have mainly been based on the species C virus Ad5, have not yet attained their expected clinical potential. This may be due to the limited efficiency of vector transduction and spread throughout tumours.
CAR down-regulation during tumour progression: implications for Ad5-based cancer gene therapy
As described above, the importance of CAR in the entry of many Ad serotypes into cultured cells is well established; however, its role in the human host is less clear. Nonetheless there is a considerable body of evidence indicating that CAR contributes to the uptake of species C Ads and therefore its expression levels need to be considered when designing efficient vectors. The expression of CAR is down-regulated in many cancers [151–155]. Lowered levels of CAR reduce entry of Ad5-based vectors to cultured cancer cells [151]. Loss of CAR appears to correlate with the aggressiveness of tumours, with the most aggressive tumours displaying the lowest levels of CAR. As CAR functions in cell adhesion, it was suggested that loss of CAR may reduce contacts between cells, increasing cellular proliferation and promoting migration, suggesting a tumour-suppressive role for CAR [152,156]. The loss of CAR has also been shown to directly influence cell migration and invasion in gastric cancer cells, contributing to tumour dissemination [157].
To circumvent the limitations imposed by reduced CAR expression, numerous strategies have been devised. While many cell proteins are down-regulated during cancer progression, there is also up-regulation of others, in particular cell-surface proteins. It is possible to exploit the up-regulation of these proteins by re-targeting Ad vectors to novel attachment molecules [158]. The incorporation of tropism-modifying motifs within fibre structures can be utilized for this purpose. For example, incorporation of an RGD-motif within the HI loop of the fibre knob redirects primary virus interactions towards integrins [159,160]. Furthermore, alterations have also been made to re-target Ad5 towards JAM-1 (junctional-adhesion molecule 1) and HSPG [161,162]. Another strategy to modify Ad5 tropism involved the incorporation of fibre knobs from different species of Ad, either human or non-human, into an Ad5 vector backbone. The use of fibre proteins from non-human Ads, such as those from mouse, dog or sheep, not only modifies the tropism, but shows reduced immunogenicity when compared with vectors based on Ad5 [163–165]. Fibre proteins from the human species B viruses are most commonly utilized in this 'pseudotyping' strategy, as these viruses interact with cells independently of CAR.
Species B Ads as gene therapy vectors
There are numerous studies in which species B fibre proteins have been incorporated into Ad5-based vectors, many of which have been based on the CD46-binding serotype Ad35. CD46 is frequently up-regulated in many types of tumour, possibly because increased CD46 expression aids tumour evasion and destruction by the complement system [166–168]. Ad5 vectors carrying Ad35 fibre knobs (Ad5/35F) have shown particular promise in the treatment of gliomas and B-cell tumours [169–171]. In addition, Ad5/35F oncolytic vectors displayed enhanced anti-tumour activity in head and neck cancer compared with Ad5 [172–174]. Ad3 is another species B Ad which has been studied for its potential use in gene therapy. Modification of Ad5 viruses to carry Ad3 fibres (Ad5/3F) has generated vectors which show greatly improved gene transfer to malignant glioma cells when compared with Ad5 vectors [76,175]. In addition, replication-competent Ad5 vectors expressing Ad3 fibre proteins have shown efficient entry and oncolysis in a panel of ovarian cancer cell lines [170,176–178]. Ad5 vectors carrying Ad3 fibre proteins have also been found to display high oncolytic potential in cell lines derived from cervical carcinoma, melanoma, prostate and renal cancers [179–182]. In addition, a replicating Ad11 vector has shown efficient transduction and oncolytic activity in human prostate cancer cells and xenografts [183]. It is likely that fibre proteins from other species B Ads will also prove useful in improving the entry of Ad5 vectors into target cancer cells.
Comparative aspects of Ad replication
Several studies have shown that fibre proteins from a number of species B Ads can be used to improve Ad5 transduction of cancer cells, suggesting that the species B Ads may be important in the development of replication-competent Ads. However, whether these viruses will demonstrate greater oncolytic activity when compared with species C viruses and warrant development as vectors in their own right remains unclear. The success of oncolytic therapy is dependent not only on the efficient entry of the viral vector to target cells, but also on the replication of the virus, as well as the production and release of infectious progeny virus which can enter and replicate in neighbouring tumour cells. Therefore, in order to establish the extent to which species B Ads may play a role in future oncolytic therapies, it is crucial that the steps involved in each of the stages of the viral life cycle be fully elucidated. This will address the question of whether greater clinical efficacy will be achieved using oncolytic viruses based on entire species B Ads or whether exploitation of species B virus tropism, for example by modifying Ad5 vectors to carry species B fibres, will prove more effective.
There are many aspects of the species B virus life cycle which have not yet been studied in depth. For example, the events regulating endosomal release of Ads, trafficking to the nucleus, nuclear import and gene expression, were determined using species C viruses. In addition, knowledge of Ad DNA replication, capsid assembly and release have all been derived from investigations with species C viruses. As described above, the receptor usage of species B Ads has previously been studied in great depth [184]. As a result, the species B viruses have been further classified into three groups related to findings based on their interactions with CD46 [184]. This division was made using anti-CD46 antibodies and soluble CD46, in addition to competition studies with labelled and unlabelled Ads and fibre knobs. Group I species B Ads were found to interact solely with CD46 and include serotypes 16, 21, 35 and 50. Group II contains serotypes 3, 7 and 14 and are proposed to interact with a receptor independently of CD46. This unidentified cell-surface molecule(s), termed receptor X, has been proposed to be a glycoprotein, expressed in many cell types, including mesenchymal and undifferentiated embryonic stem cells [184]. Finally, Ad11 was assigned to Group III as it was capable of utilizing both CD46 and receptor X for cell attachment. In agreement with this new classification, a recent study reported similar groupings of the serotypes using haemagglutination of CD46-expressing RBCs (red blood cells) derived from transgenic mice [185]. Serotypes 11p, 34, 35 and 50 were found to agglutinate CD46-expressing RBCs but not control CD46-negative non-transgenic RBCs. Ad16 and Ad21 demonstrated incomplete haemagglutination with RBCs expressing CD46, with no agglutination of control RBCs, whereas serotypes 7 and 14 did not agglutinate CD46-expressing RBCs. Interestingly, Ad3 was found to agglutinate control and CD46-expressing RBCs with similar efficiency, suggesting that Ad3 interactions with these cells were independent of CD46.
As CD46 is often up-regulated in cancer cells, there is an obvious advantage in developing a CD46-binding oncolytic agent. However, the expression of CD46 on all nucleated cells may prove problematic for the specific targeting of viruses which demonstrate a high affinity for this complement receptor. Nonetheless, the species B Ads include a number of serotypes which do not interact with CD46 and there is evidence to suggest that these non-CD46-targeting species B Ads may possess greater therapeutic potential than viruses which utilize CD46. Serotypes 3, 7, 11 and 14 have all been found to interact more efficiently with cancer cell lines compared with primary cells, suggesting that receptor X, like CD46, may be found at higher levels in tumours compared with normal tissues [184]. Tumour sections from ovarian cancer patients and mouse xenografts have also been shown to display an epithelial phenotype in which the cells are polarized and CD46 is located within tight junctions [186]. The distribution of CD46 on these cells makes it inaccessible to CD46-targeting Group I species B Ads. In comparison, serotypes 3, 7 and 14, which do not target CD46, were found to be much more efficient in killing these epithelial ovarian cancer cells. As most cancers are derived from epithelial cells and have an epithelial-like phenotype, it seems likely that Group II Ads will be important for future vector development. However, the identification of cell-surface attachment sites and receptors used by these viruses to promote entry will be essential in establishing the full potential of these viruses.
Clarification of a role for integrins in the uptake of species B Ads to host cells will also improve understanding of the entry pathways of these viruses. Although integrins have been found to be present within Ad3- and Ad35-induced macropinosomes, a direct function for these cell-surface molecules has not been confirmed. As cross-linking of CD46 at the cell surface by a ligand is sufficient to stimulate uptake of this protein by macropinocytosis, it seems unlikely that the uptake of species B Ads would require a secondary interaction. However, it is possible that such an interaction may improve the efficiency of uptake [95]. Interactions with integrins were found to be important for Ad35 entry to haematopoietic cells [187]. Conversely, it has been suggested that Ad35 vectors utilize an integrin-independent pathway for entry into airway epithelial cells [188]. Thus interactions with integrins by the species B Ads may be serotype- and/or cell-type-dependent.
The interaction of the Ad capsid with blood factors and neutralizing antibodies is also an important consideration when developing these viruses as therapeutic agents. While vectors can be delivered into tumours by direct injection, some cancers cannot be targeted in this manner and vectors must be introduced intravenously. Unfortunately, the majority of the population have antibodies against Ad5 fibre and hexon proteins which could significantly reduce the efficacy of vector delivery in such individuals. In contrast, several of the species B Ads display a much lower seroprevalence than Ad5 and may therefore be more amenable to intravenous administration [189,190]. In addition, the high affinity of Ad5 hexon for coagulation factors may lead to liver sequestration following delivery to the bloodstream, again reducing vector delivery to target cells. Many species B Ads also show a lower affinity for the blood coagulation factor FX compared with Ad5. Ad35 vectors, for example, exhibit a 10-fold lower affinity for FX compared with Ad5 [191]. In addition, CAR has recently been identified on human erythrocytes, which may limit the targeting efficiency of CAR-binding vectors [192]. These factors suggest that certain species B viruses may demonstrate improved efficacy and safety in vivo compared with Ad5, which supports further investigation of species B Ads as whole vectors rather than simply contributing the fibres in a hybrid Ad5 vector.
The impact of FX on the liver tropism of intravenous Ad5 vectors has been well documented. However, there is evidence to suggest that blood factors can also promote the uptake of Ad into certain epithelial and tumour cell lines, which may indicate that these factors can influence interactions with a broader range of cell types [193,194]. Furthermore, it has been proposed that vitamin-K-dependent zymogens may also function outside of blood, as extravasation of plasma proteins to the apical side of airway epithelial cells has been reported [195]. In addition, Factor VII, FX, fibrinogen and Factor XIIIA have all been found to be produced and secreted by respiratory epithelial cells [196]. These findings suggest that blood factors could also contribute to Ad interactions during normal infection, allowing species C viruses to transduce airway epithelial cells independently of CAR [194]. It is also possible that other host factors may have an impact on Ad interactions with host cells. For example, DPPC (dipalmitoyl phosphatidylcholine), which is secreted by alveolar type II epithelial cells and Clara cells, has been shown to enhance Ad2 and Ad5 infection in rabbit and rat lungs, as well as improving gene transfer to A549 cells, independently of CAR and integrins [197,198].
Lactoferrin is another important example of a body fluid component which can mediate Ad interactions with host cells. Lactoferrin generally exhibits antiviral activity either through the direct binding of viral capsid components or through interaction with cellular receptors used by viruses [199]. However, in a study to explore the role of lactoferrin in Ad37 eye infections, it was unexpectedly found that lactoferrin enhanced the entry of Ad5 to both ocular and respiratory epithelial cell lines [200]. Lactoferrin was proposed to bind to the surface of the virus and form a bridge to unidentified receptors on the surface of epithelial cells. Bovine lactoferrin was also found to promote Ad5 interactions with DC-SIGN (dendritic cell-specific intracellular adhesion molecule-3 grabbing non-integrin) expressed on the surface of dendritic cells [201]. The entry of recombinant Ad35 vectors to dendritic cells was also found to be enhanced by human lactoferrin via CD46 receptors [201]. Interestingly, enhancement of infection of respiratory and ocular cell lines by lactoferrin was not found for species B serotypes 7 and 11 suggesting a possible serotype- and cell-type-specific role for lactoferrin in Ad uptake [200]. The possibility that 'bridging' factors could play a role during Ad infection is an interesting and largely unexplored aspect of the Ad life cycle.
More relevant human cell/tissue models are required in order to study Ad–host cell interactions. It has been noted, for example, that Ads do not achieve nuclear localization during infection of primary cells, such as fibroblasts and endothelial cells, yet staining of the nuclear periphery has been observed following Ad infection of transformed cell lines [30]. This might suggest a limitation in the use of cell lines to establish detailed aspects of Ad interactions with the host, as findings may not be representative of those that occur in primary tissues. It is also clear that cultured cell lines lack the three-dimensional cell–cell and cell–matrix contacts present in tumours, and therefore offer limited information on the spread of oncolytic viruses through the tumour mass [202]. The use of tissue slices from human cancers and non-target organs may therefore represent a more appropriate model system for studying Ad–host cell interactions, as well as for the development of selectively replicating Ad vectors [203]. Such a system may also be useful in addressing whether complete species B viruses or Ad5 vectors carrying species B viruses show greater oncolysis. The apparent lack of an Ad death protein with which to facilitate cell lysis suggests that the species B viruses may be released from cells more slowly than species C viruses. This is supported by early plaque assay studies which show much smaller plaque morphology in KB cells infected with species B Ad serotypes (3, 7, 11, 14, 16 and 21) compared with Ad2 and Ad5 [204]. This might suggest that Ad5 vectors carrying heterologous fibres may show greater clinical benefit. Alternatively it may be that species B viruses modified to express the ADP will display superior oncolysis. However, the precise strategy used by species B viruses to facilitate cell lysis and release remains to be determined and could prove significant to the development of the viruses as oncolytic vectors. It will also be interesting to establish a role for the unidentified putative protein assigned to the L6 region of certain species B Ad genomes [205,206].
It is evident that a number of questions remain concerning many aspects of species B Ad entry and replication. As vectors based on species C Ads do not necessarily demonstrate efficient transduction of cancer cells, there is a need to develop vectors from alternative human (and perhaps non-human) Ad species. The species B Ads appear to offer a number of advantages compared with current vectors, but whether these features will translate to clinical success has yet to be determined. It will be important to assess whether vectors based on entire species B Ads will demonstrate greater oncolytic activity compared with Ad5 viruses pseudotyped with species B fibres. This will require detailed analysis and comparison of each of the steps involved in the life cycle of both species B and species C viruses. A comparison of the utility of species B viruses which target CD46 compared with those which use receptor X will also be important. The serotype chosen for a particular application may be dependent not only on the target cells, but also on the route of vector administration. Ultimately, a more detailed understanding of the molecular biology of species B Ads will lead to the development of safer, more efficient and more effectively targeted gene therapy vectors.
- Ad
Ad death protein
coxsackie and adenovirus receptor
Chinese-hamster ovary
chromosome region maintenance 1
- cryo-EM
cryo-electron microscopy
- CtBP1
C-terminal binding protein 1 of E1A
- dsDNA
double-stranded DNA
Factor X
heparan sulfate proteoglycan
microtubule-organizing centre
nuclear pore complex
p21-activated kinase 1
protein kinase C
prostate-specific antigen
- Rb
red blood cell
short consensus repeat
surface plasmon resonance
terminal protein
We thank Mark Parsons and James Findlay for help and advice in the preparation of this paper.
Research in our laboratories is funded by Yorkshire Cancer Research and the Biological Sciences and Biotechnology Research Council.


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Zakir Hussain Actor 2013 Horoscope
Name: Zakir Hussain Actor
Date of Birth: Friday, March 09, 1951
Time of Birth: 11:00:00
Place of Birth: Mumbai
Longitude: 72 E 50
Latitude: 18 N 58
Time Zone: 5.5
Information Source: Kundli Sangraha (Tendulkar)
AstroSage Rating: Accurate
Zakir Hussain Actor 2013 Horoscope
Year 2013 Summary Horoscope
This a good position for Muntha. Zakir Hussain Actor's intelligence will help Zakir Hussain Actor earn accolades from various quarters of life. Zakir Hussain Actor might shine brightly in profession and business. Birth of a child in the family will bring happiness and joy for Zakir Hussain Actor. This period is characterized by wisdom and religious learning for Zakir Hussain Actor. Zakir Hussain Actor will have visit to the religious places or the places of recreation. Zakir Hussain Actor will get honor and appreciate from rules and higher authorities.
Mar 9, 2013 - Apr 27, 2013
There will be a strong influence from others to help Zakir Hussain Actor create more personal security in having Zakir Hussain Actor's material needs met. Money will definitely be coming Zakir Hussain Actor's way and will greatly influence Zakir Hussain Actor's personal beliefs, dreams and philosophies. Zakir Hussain Actor will get recognition of Zakir Hussain Actor's merits by the government and higher authorities. Zakir Hussain Actor have a friendly nature, and feel very comfortable enjoying the group dynamics of different social scenes; Zakir Hussain Actor may get disturb a bit due to health ailment. Personal transformation is far more appealing than outer changes.
Apr 27, 2013 - Jun 23, 2013
The period opens with a difficult phase in career. A dip in activity & opportunity will be experienced, although there could be rise in irrelevant activity in career. New investments or risky deals should be avoided as there could be chances of losses. It will not be advisable to commence new project or make new investments. Zakir Hussain Actor should avoid getting aggressive with Zakir Hussain Actor's superiors. It would be better to use Zakir Hussain Actor's own skill and caliber other than thinking of taking help from others. Possiblity of theft or loss of money due to some other means is also there. Take proper care of yourself and Zakir Hussain Actor's family members. Zakir Hussain Actor may also receive bad news regarding some one's demise.
Jun 23, 2013 - Aug 14, 2013
This is not a very satisfactory period for Zakir Hussain Actor. Zakir Hussain Actor may get indulge into sudden losses financially. Failure in attempts will make Zakir Hussain Actor feel frustrated. Zakir Hussain Actor will have to slog as the work burden will be too much. Family life will also create tensions. Don't try to take risks in business matters as period is not very harmonious to Zakir Hussain Actor. Zakir Hussain Actor's enemies will try to tarnish Zakir Hussain Actor's image. Zakir Hussain Actor may also get indulge into unnecessary expenses. Health may also give some trouble. Especially old people may encounter catarrh and phlegmatic troubles.
Aug 14, 2013 - Sep 04, 2013
If in a job, the year will begin very aggressively .There will be dynamism & growth. However work environment will remain stressed and there could be controversy & issues with superiors. In general this period is not very good as very close associates, friends and family members may appear distant. Not much change is expected or recommended. Zakir Hussain Actor's attitude and habit of using foul language may create tensions with Zakir Hussain Actor's near and dear ones. Therefore, try to control Zakir Hussain Actor's words.
Sep 04, 2013 - Nov 04, 2013
In this period Zakir Hussain Actor will spend more on luxuries and comforts but it would be better if it is checked out. Zakir Hussain Actor may face disappointments in love affairs and trouble in family life. Zakir Hussain Actor's rivals will try to harm Zakir Hussain Actor in each and every possible way so try to be more concerned when dealing with any kind of personal or professional matters. A worry related to health of Zakir Hussain Actor's family members is on the cards. Though financially, it is not a bad period but still Zakir Hussain Actor should put hold on over expanses. Get proper care of Zakir Hussain Actor's own health.
Nov 04,2013 - Nov 22, 2013
Don't try to be aggressive in nature because Zakir Hussain Actor's aggressiveness can shove Zakir Hussain Actor into difficult situations. There will be difference of opinion, quarrels, and fighting with Zakir Hussain Actor's friends. So, try to maintain good relations otherwise there is possibility of straining relations with them. There will be ups and downs financially. Lack of harmony and understanding is indicated in family life. There are possibilities of distress from spouse and mother. Care must be taken regarding health. The diseases that require immediate attention are headace, eye, abdominal disease, and swelling of the feet.
Nov 22, 2013 - Dec 23, 2013
This is a period of financial stability for Zakir Hussain Actor. During this period Zakir Hussain Actor can work over Zakir Hussain Actor's hopes and ambitions and give them a better shape. This is a favorable time for love and romance. Zakir Hussain Actor will develop new friendship which will be very rewarding and helpful. Zakir Hussain Actor will enjoy respect and honor from learned people and will be quite popular with the opposite sex. Long distance travel is also indicated.
Dec 23, 2013 - Jan 13, 2014
This is the time which will show Zakir Hussain Actor mixed results. Don't overlook minor health related problems because it may turn into a big one. Some diseases that require special attention are ulcer, rheumatism, vomiting, head an eye troubles, pain in joints or injury due to falling of heavy metallic lump etc. Difficult situations may arise in Zakir Hussain Actor's path but don't lose Zakir Hussain Actor's heart in the adversities as Zakir Hussain Actor's self-confidence will work in Zakir Hussain Actor's favor. Dispute with government and senior officials are on the card, so Zakir Hussain Actor are advised to be careful. This is not a favorable time for speculation or taking risks.
Jan 13, 2014 - Mar 09, 2014
New investments & risks should be totally avoided. There could be hurdles & hassles during this period. If working as a professional, the year will see progress, but only if Zakir Hussain Actor work hard and keep a long term & stoic attitude. There would not be no shortcut to success. Zakir Hussain Actor should work on keeping a stable and steady nature for better results. Work environment could be stressed and erratic as the year opens. Zakir Hussain Actor should avoid new progress or high activity during the period. Health problems may not permit Zakir Hussain Actor to keep Zakir Hussain Actor's promises on the positive side of this period. Health needs a check and problems due to fever is most likely.
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Photo Credit: University of Kentucky Athletics
In the recent MLB draft, the Red Sox selected plenty of high powered offense. The former Wildcat will provide plenty of that in Boston.
The Boston Red Sox selected C/1B Kole Cottam with their 4th round pick. The prolific hitter had a solid season at the University of Kentucky compiling a .352 average with 77 hits, 19 homeruns, and 51 RBI's.
Cottam's strong season garnered 1st team All-SEC honors and 2nd team All American status.
Off the field, Cottam has overcome repeatedly. The Red Sox draftee has suffered through 13 surgeries on his right eye due to a congenital melanocytic nevus. This is a rare condition that is present in 1% of infants.
We had the opportunity to catch up with the Sox 4th round pick!
Question: Pre-Draft: Who was your favorite MLB team?
KC: With my dad growing up just outside Toronto the Blue Jays have always been my favorite team growing up.
Question: What player did you idolize growing up?
KC: My favorite player was Buster Posey because of the way caught and hit at such a high level.
Question: Is there one pitcher in the big leagues that you are looking forward to facing?
KC: I think growing up and seeing the dominance of elite pitchers like Justin Verlander, it would be pretty cool to face him!
Question: Who do you model your game / approach after?
KC: I try and model my approach offensively after Evan White (former UK teammate and member of the Seattle Mariners) and my catching after Posey as well.
Question: Did you see the Red Sox as a fit before the draft? If so, why?
KC: Prior to the draft I thought the Red Sox would be a great fit because of their great player developers and their success of developing their own guys. And who wouldn't want to play for the Red Sox! They're such a storied franchise with a history of success.
Question: What guy are you most excited to play with in Boston? This can be a minor league, or major league level player
KC: I think I'm really excited to meet Mookie Betts. Both being guys from Tennessee, and with his success, it will be really exciting to meet him.
Question: You've played both catcher and pitcher at Kentucky, have the Red Sox given you any indication of which route they'd like you to pursue? If not, do you have a personal preference?
KC: I love catching and they want me to stick behind the plate as well!
The most intriguing takeaway from the interview is the fact that the Red Sox are looking to keep Kole behind the plate. The Sox catchers have struggled with offensive output over the past few years – an issue that many fans have noticed. He can very easily develop into a 20+ homerun bat.
The Red Sox have very minimal catcher depth within the organization as Roldani Baldwin is the only catcher listed within their Top 30 Prospects. Cottam figures to slide into the organization's top 30 and has plenty of upside if his defensive ability matches his raw power.
Cottam has been assigned to the Low A, Lowell Spinners to begin his Major League career.
If you're able to get to a Spinners' game, be sure to head to batting practice – he can put on a show!
Your new SEC HR leader…@UKBaseball's Kole Cottam pic.twitter.com/hYuQh4EUoX
— SEC Network (@SECNetwork) May 22, 2018


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Hi there!
When I first heard there was a white deer in the woods of my hometown Bochum, I got ants in the pants, took my digicam and went backpacking. Very soon I noticed that the deer which I was looking for wasn't there anymore. That was quite disappointing to me, since I really wanted to see it. So I went backpacking in the forests and reserves of various towns in North Rhine-Westphalia until I eventually found a white deer on Wednesday. Of course, I took pictures which I would like to share with you now.
Isn't it majestic and awesome?
Looking a little further I even met its offspring. It embraced my presence, so I could approach it. Enjoy:
What a peaceful and calm expression.
Take care, friends.


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Twilight Honor was an adventure book for the Legend of the Five Rings roleplaying game. It was the second of the Shadowlands 'S' series of adventures, designed to help Game Masters integrate the deadly potential of the Shadowlands into their campaign, without tearing down the Kaiu Wall.
Credits Edit
- Written by: Jim Long
- Game System by: David Williams and John Wick
- Art Director: K.C. Lancaster
- Cover Artwork: Cris Dornaus
- Interior Artwork: Cris Dornaus
- Maps: Cris Dornaus
- Artwork Prepress: Steve Hough, Brendon Goodyear
- Line Editor: D.J. Trindle
- Editing: Ree Soesbee, D.J. Trindle
- Interior Layout: Brendon Goodyear
Table of Contents Edit
Introduction (page 4) Edit
- Gamemaster Notes.
Part I: A Simple Task (page 7) Edit
- Now, What?
Part II: Hida Palace (page 9) Edit
- The Crab Captain
- The Young Hero
- Another Chance?
Part III: Kuni castle (page 13) Edit
- The Road North
- Shiro Kuni
- Life in the Castle
- Making a name for yourself
- Hiruma Makasu
Part IV: Into the Shadowlands (page 19) Edit
- Courtiers
- Shadowlands Weapons
- Ambush
- A Long Walk Home
- Delaying the Inevitable
- A Gallant Defense
- Medic!
- Life in the Castle
Part V: You Want Us to Go Back? (page 27) Edit
- A Difficult Situation
- Going with Shiroi
- A Bad Idea
- The Lesser of Two Evils
- The Other Side of the Ambush
- Haikyo no Hiruma
- Back to the Castle
Part VI: Defense of a Clan (page 35) Edit
- All Quiet in the Kaiu Wall
- Twilight Battle
- The Great Bear
- The Battle for Kuni Castle
- The Aftermath
Consequences and Rewards (page 44) Edit
Dramatis Personae (page 48) Edit
- Togashi Yokuni
- Hida Yakamo
- Hida Shiroi
- Hiruma Makasu
- Hida Kisada
- Tonsu
- Daidoji Jinshi
- Muago
Community content is available under CC-BY-SA unless otherwise noted.


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We provide a huge number of options for facial animation. We can work with audio, video, multi-camera data and depth sensors. In many cases, this data is captured at the same time as body motion data, but sometimes it's not possible to capture every performer on a motion-capture stage. However, this shouldn't stop you considering great facial capture during voice-recording. The best audio studios (such as Side, London - where this clip was recorded) can supply facial capture hardware and services. This data can be rapidly processed by Cubic Motion. Here we show the draft result as a simple Maya playblast.
A common concern is head motion. Ultimately, the facial performance will be placed in a scene, which will to some extent determine the required head movement. However, facial movements are an integral part of performance, and those movements picked up during voice recording time can really add to the expressiveness of a result. Cubic Motion provides head motion data from a reference camera (free-of-charge on large projects) alongside the animation from the head-mounted camera. This motion can be used wholly or partly in the final integrated result.
This simple but effective solution ensures that the face itself is always captured with maximum integrity whilst also providing head motion. We believe this is far superior to methods which try to capture facial detail and head pose from a single device, where the integrity of facial capture is likely to be compromised by pose changes.
Shout out to 3lateral - creators of the "Lydia" rig shown in this playblast.


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Cheers! oneworld is best alliance for in-flight wine again
Judges at a leading international wine-tasting competition have raised their glasses to oneworld® as the best airline alliance for the wines served in-flight by its member airlines for the ninth year in a row.
The Cellars in the Sky Awards, now in their 32nd year, are run by Business Traveller, the multi-national magazine group, in association with the International Wine Challenge. The 2017 awards were presented last night at a ceremony at London's historic Trinity House, following blind tastings by a panel of four leading experts of hundreds of wines served on longhaul flights by 32 airlines from around the world.
This latest accolade consolidates oneworld's position as the world most highly prized airline alliance, as the current holder also of six of the leading overall "best airline alliance" awards presented internationally, along with the only other alliance award presented for the quality of wines offered by airlines, Global Traveler's Wines on the Wing.
oneworld's member airlines also flew high in the Cellars in the Sky 2017 awards.
Qantas received three Gold medals – for Best First Class White, Best First Class Cellar and Best Business Class Fortified/Dessert Wine.
Cathay Pacific and Malaysia Airlines jointly shared the Gold Medal for Best First Class Sparkling.
American Airlines, British Airways, Finnair, Japan Airlines, Qatar Airways and SriLankan Airlines were also honoured with Silver and Bronze Medals.
The judges included luminaries from the world of wine, led by co-chairman of the International Wine Challenge Charles Metcalfe.
For the results in full, see businesstraveller.com
Last year, oneworld member airlines served some 30 million bottles of wine, including around 2.5 million bottles of Champagne.
Among wine and beverage fun facts from across the alliance:
- Qantas is the third biggest buyer of wine in Australia, selecting from 150 plus Australian wine makers.
- British Airways offers a choice of three Champagnes and seven different wines to First Class passengers – and five wines in Club World (longhaul Business Class).
- Many oneworld member airlines offer their own alcoholic and non-alcoholic signature cocktails.
- Cathay Pacific has brewed its own signature beer – Betsy – specially crafted to be ideal at 36,000 ft.
- British Airways commissioned its own signature gin.
- oneworld member airlines focus also on non-alcoholic beverages. Among them, Finnair served a million litres of blueberry juice, Finland's signature drink, while Qantas serves barista coffee in most of its lounges.
Other "best airline alliance" awards currently held by oneworld include:
- FlightStats' Airline Alliance On-Time Performance Service Award for 2017, for the fifth year running.
- Global Traveler magazine's 2017 GT Tested Reader Survey – for the eighth year in a row.
- Business Traveller's 2017 Best Airline Alliance for the fifth consecutive year.
- Business Traveler North America's 2017 Best in Business Travel Awards, for the third year running.
- World Travel Awards' World's Leading Airline Alliance 2017 for the 15th year running.
- Trazees' Favorite Airline Alliance, for the second time.


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Welcome to Thunderbird Add-ons.
Add extra features and styles to make Thunderbird your own.Close
Toggle Word Wrap 2.0 Requires Restart
by Jan Kiszka
Toggle word wrapping and <PRE> element wrapping in the message composition and HTML display windows, respectively. Based on original version by Kaspar Brand.
About this Add-on
Unchecking Enable Word Wrap in the Options menu (or pressing the keyboard shortcut) will temporarily turn off word wrapping for the current message - lines will take up the whole width of the composer window, and no line breaks will get added to the outgoing message (with the exception of "hard" line breaks inserted manually).
Checking Enable Word Wrap will enable word wrapping again and set it to the standard width (which is modifiable through the mailnews.wraplength preference in the Config Editor, the default is 72 characters).


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As a volunteer-run event Hibanacon relies on the generosity of its attendees to make it a success. There are only so many places the committee can be in at once, and from time to time we need a hand. If you can spare a couple of hours over the weekend to help Hibanacon it's very much appreciated.
What you might be helping with:
We'll provide briefings for each task and give you a gopher badge so you'll know what do, and look official doing it. Some drinks and snacks will be available in Ops for volunteers there will also be an end of event party on the Sunday evening.
If this sounds interesting then once you've bought a ticket to Hibanacon, and had a look at our Gopher code of conduct please email email@example.com answering the below questions, or come along to ops at the event and ask about gophering.
Badge Name
Relevant experience: gophering, team leading, tech knowledge etc


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Home » Fairies & Friends » Fiddlehead Fairies » Tiny Fairy Rosie
Tiny Fairy Rosie resembles a blooming rose garden from head to toe. At only 1.50-inches high, she is not much larger than a rose bud. Her chocolate colored hair is pulled back in a prim and proper coif with a blooming rose accenting the side. Three ruby red rose blooms form a necklace across the tiny neckline of her rather formal dress. The skirt of Rosie's dress is full and pleated with the appearance of a poufy crinoline underneath. Tiny Fairy Rosie will add a burst of rose-colored hues to the smallest miniature garden setting.


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I'm A Celebrity… Get Me Out Of Here! 2014 campmates reveal their phobias
Ten celebrities are about to head into the jungle to face all manner of creepy crawlies, dark nights and Bushtucker Trials. Once again, they've gone and told us what they're afraid of…
Every year the I'm A Celebrity campmates head into the jungle and are very honest about what they're afraid of. Haven't they learned? We all get on the phone and vote them into the very trial that contains their worst nightmare (we're a mean bunch. They enjoy the sun, we enjoy the power).
So as they pack their bags and head into camp, here's what each celebrity is really not looking forward to…
Michael Buerk
Hang on, we've got a sensible one straight out of the gate. "There is no point saying you hate spiders as the next thing you know, you will be banged up with them!" says the newsreader. "I am allergic to cats, but there aren't any in the jungle!"
On joining the show he says he's a "frightful snob and perhaps it's time for me to take myself less seriously". But he's going to miss booze. "I am not sure I have been three weeks without a drink since I was a teenager!"
Jimmy Bullard
"I hate snakes, spiders, all the animals you find in there." Oh dear, this is what we were talking about Jimmy. "I am petrified of animals and I am determined not to refuse the trials. But there again, if lasagna is put in front of me rather than a cockroach, then it's going to be hard to turn the lasagna down."
But the former Wigan player says he's looking for a new challenge to fill the football void and says, "There won't be any boring times as I will make sure there isn't."
Craig Charles
The Coronation Street actor say he hasn't got any phobias but "doesn't like the look of" the eating trials. "I turned 50 this year and I thought I should do it now while I can still parachute in!"
And Craig is expecting this year to be harder. "Last year was a well fed show and I know they are going to make it tougher. A hungry camp is an exciting camp!"
Gemma Collins
Gemma's got a few worries: "Claustrophobia, every single creepy crawly. I just have to see an animal and I jump!"
The TOWIE star adds, "Every time I think about going in the jungle I get very, very nervous. I get stomach cramps, I feel sick. Even talking about it scares me.
"I've only got to see an animal on TV and I start screaming… this is all completely out of my comfort zone, I do however believe you have to grow in life," Gemma adds. Hopefully Joey Essex talked to her about "confrontating" her fears, eh?
Carl Fogarty MBE
"I am not fond of heights, but lack of food, boredom and people are the three things I hate most," says one of the world's legendary motorbike drives. Oh, oh. We're ready for the fireworks as he adds, "I am not a people person. I like my own company and I like my own space."
"There is a big part of me that is saying 'You can't do that' but I am definitely at a stage in my life where I want to give it a go."
Nadia Forde
Top of model and singer Nadia's list of fears are "creepy crawlies, heights, the dark. I've made no secret of my fears!" But Nadia has a plan. "It's a case of mind over matter and I am going to have to imagine I am not in my body when I am doing it all! You are going to a situation where you are not doing it for yourself. You are part of a team."
And it seems Nadia will keep the team cheery. "I can chat all day long. I do like hearing stories. I will be talking to people and who knows, maybe I will conduct a sing-song around the campfire!"
Vicki Michelle MBE
'Allo 'Allo actress Vicki hopes she won't be saying hello to any snakes and spiders. "My daughter is scared of spiders and I have always said that I must be braver than her. I do think you are a braver person as you get older."
Although she adds, "Having them all over you will be a different ball game. They are, however, only creatures – and I need to get that into my head." Convinced? We hope she is!
Tinchy Stryder
Another one who knows how to play the game already, Tinchy says, "I don't want to say anything to jinx myself." The singer, who recently released a charity single with the Chuckle Brothers says, "I like challenges. I like doing things you don't expect from me. It's going to be really nice for people to see me as me."
Melanie Sykes
"Heights, claustrophobia – all that is required out in Australia" is worrying this TV presenter. Hopefully her co-star and former jungle campmate Gino D'Acampo has given her some tips.
"It will be a testing time for me," she admits, adding "But I know myself well enough not to put myself in a situation I can't handle.
"I don't like being hungry, but it is an experiment and it's a great adventure."
Kendra Wilkinson
Kendra is in for a shock as she reveals she's never watched the show before. "People are telling me about it".
"I decided I wanted to do it as I needed a shake up in my life," she adds but says she's a "complete wuss".
"You are all in for a treat. I don't know what I am capable of doing but I am afraid of free falling. I quit Splash! because I couldn't dive off a 3m board. I am afraid of bugs and snakes but I can't tell you yet if I have a phobia of them. I do think I am brave to have even signed my name on the papers [of the show]! It's going to be a case of mind over matter."
I'm A Celebrity… Get Me Out Of Here! returns Sunday 16th November at 9:00pm on ITV
PLUS: we want you to vote for your favourite jungle winner in our I'm A Celebrity champion of champions poll


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Global Warranty Management System Market 2015 - Global Forecast to 2020 with IBM, Oracle, Pegasystems, PTC & SAP Leading the $3.4 Billion Market
04 Jan, 2016, 16:10 GMT
DUBLIN, Jan. 4, 2015 /PRNewswire/ --
Research and Markets (http://www.researchandmarkets.com/research/nwmrjl/warranty) has announced the addition of the "Warranty Management System Market - Global Forecast to 2020" report to their offering.
The warranty management system market size is estimated to grow from USD 1.7 Billion in 2015 to USD 3.4 Billion by 2020, at a Compound Annual Growth Rate (CAGR) of 14.4% from 2015 to 2020
In the milieu of automating all service management processes, ineffective claim processing and valuation of warranties lead to the increasing need for warranty management systems. Most organizations are unaware about the impact of insufficient and lagging product criticism, ineffective repair management, hampered customer brand loyalty, and increased dissatisfaction among customers. With the convergence of analytics and warranty intelligence, the market has a huge opportunity to grow during the forecast period.
The warranty management software solutions effectively automate the warranty management process which includes warranty coverage, administrating campaigns and product registrations, contract management, warranty and contract tracking, part return and supplier cost recovery. The service contract software segment is expected to hold the largest market share and dominate the market from 2015 to 2020. The main driver encouraging organizations to install service contract are the revenue leakages due to the use of paper documents, unstructured files, and singular systems.
Warranty intelligence software is expected to grow at the highest rate in the warranty management system market during the forecast period. The software helps to track error patterns and project accurate warranty trends, thereby saving 2-3% of the claims cost that the company spends in claims settlements. Claim management software is also one of the software solutions holding a major market share, as it develops a seamless claim processing infrastructure, which automates the process of claim processing and reduces duplication and fraud.
The major vendors in this market include IBM Corporation, Oracle Corporation, Pegasystems, Inc., PTC, Inc., SAP SE, and others.
Key Topics Covered:
1 Introduction
2 Research Methodology
3 Executive Summary
4 Warranty Management System Market: Premium Insight
5 Warranty Management System Market: Overview
6 Warranty Management System Market: Industry Trends
7 Warranty Management System Market Analysis, By Software
8 Warranty Management System Market Analysis, By Service
9 Warranty Management System Market Analysis, By Deployment
10 Warranty Management System Market Analysis, By Vertical
11 Geographic Analysis
12 Competitive Landscape
13 Company Profiles
- Astea International, Inc.
- Industrial and Financial Systems (IFS) AB
- Infosys Limited
- International Business Machines (IBM) Corporation
- Oracle Corporation
- PTC Inc.
- Pegasystems Inc.
- Tavant Technologies, Inc.
- Tech Mahindra Limited
- Wipro Limited
For more information visit http://www.researchandmarkets.com/research/nwmrjl/warranty
Media Contact: Laura Wood , +353-1-481-1716, firstname.lastname@example.com
Related Links
SOURCE Research and Markets


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Customer Service – Retail
The primary focus of the programme is to support students into the workforce after completion of the programme. It is also to support learner into further education relating to increasing the service industries (Retail and Hospitality) skills and qualifications to support entry into the workforce at a later stage.
The Customer Service programme is a training and job placement programme that links skills training and support with work experience training focused toward job placement. Each step forward has success with an end focus firmly set on employment. This incorporation of on-job industry relevant training through stair casing students into in-work support options aims to assist students to complete qualifications started in the training centre and transferring learning into a work environment.
Training with Tree House has been designed around individual training plans where learners can work through the subjects and training that will benefit them within a workplace. An understanding of management, teamwork and work ethics provides students with an insight into the everyday workings of a workplace. We incorporate a literacy and numeracy component in our training with a focus on workplace applications in a retail or distribution environment. We aim to provide education opportunities to assist with developing long term careers and personal independence.
The other main areas of skill development in the programme content include basic customer service and knowledge of health and safety practice, routine calculations, products, and legislation. In addition, the learner will be able to demonstrate a range of skills through the elective unit standards including selling, merchandising, stock control and data entry.
The educational component is designed to allow students the ability to achieve each certificate with "manageable" rates of learning and regular achievements.


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BlackPink Slays the US Billboard Charts
The YouTube view rate of BlackPink's new track "DDU-DU-DDU-DU" has already surpassed 100M, a reflection of how the popularity of these four girls are skyrocketing all over the world.
As if we need more evidence that BlackPink has the potential to be the best K-Pop Girl Group, as "DDU-DU-DDU-DU" ranks No. 55 on the Hot 100 charts, making them the second ever K-Pop Girl Group to enter the charts, breaking the record of Wonder Girls' "Nobody", which ranked No.76 nine years ago.
Who's excited about BlackPink's result?!


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This blog is mine, my thoughts, my moods my musings. Comments are welcome and un-moderated however vile comments, verbal abuse of others, will be removed.
Friday, 6 November 2009
Nothing More Need Be Said !
2 comments:
- subrosa said...
LotF, when the lads/lasses are brought back to the UK, is their coffin carried by their regimental pals or a special kind of 'undertaker' section of the logistics corp for example? Strange question to ask I know, but I heard someone say something about it this week and I missed the answer.
- 6 November 2009 at 11:35
- The Last Of The Few said...
Of all the repatriation ceremonies I have attended it is the task of the regiment of the service person.
Task is an incorrect word I hasten to add I would be honoured to carry for a colleague.
However for the case of the RAF usually that falls on the RAF regiment.
The same for the Marines and usually the regiment of the departed.
However certain regiments sizes or specialist units use a ceremonial undertaker as you say.
Most individuals when on theatre duties are officially "attached" to a parent regiment.
So you can see how it unfolds. So you are correct on both counts.
- 6 November 2009 at 12:03