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Review ARTICLE
Autophagy and immunity – insights from human herpesviruses
- School of Cancer Sciences, University of Birmingham, Vincent Drive, Birmingham, UK
The herpesviruses are a family of double-stranded DNA viruses that infect a wide variety of organisms. Having co-evolved with their hosts over millennia, herpesviruses have developed a large repertoire of mechanisms to manipulate normal cellular processes for their own benefit. Consequently, studies on these viruses have made important contributions to our understanding of fundamental biological processes. Here we describe recent research on the human herpesviruses that has contributed to our understanding of, and interactions between, viruses, autophagy, and the immune system. The ability of autophagy to degrade proteins located within the nucleus, the site of herpesvirus latency and replication, is also considered.
In mammalian cells three different autophagy pathways have been shown to deliver cytoplasmic proteins or organelles into the lumen of lysosomes for degradation. The first pathway, microautophagy, is a process in which the lysosomal membrane invaginates, capturing portions of cytoplasm. Endosomal microautophagy has also recently been described (Sahu et al., 2011). The second pathway, chaperone-mediated autophagy (CMA), involves the transport of proteins containing a specific motif into the lysosome via the concerted actions of chaperone proteins and the lysosomal membrane protein LAMP2a (Dice, 2007). The third pathway, macroautophagy (hereafter referred to simply as autophagy) is the best described of the three and can be divided into six sequential steps: initiation, nucleation, elongation, closure, maturation, and degradation. Each step is a highly regulated process involving the coordinated action of numerous autophagy-related (ATG) and other proteins. Initiation of autophagy occurs upon activation of the ULK1 and UKL2 complexes following release of their inhibition by the mTOR complex. Nucleation defines the site for the autophagic membrane to begin to form in the cytoplasm and depends on the oligomerization of Beclin-1 that acts as a platform for recruitment of multiple other autophagy-related proteins. Elongation of the autophagic membrane around a portion of cytoplasm involves the recruitment of more proteins and lipidation of the microtubule-associated protein light chain 3 (LC3) by the E2-like enzyme ATG3. Closure of the membrane completes the formation of the double membrane vacuole, called an autophagosome, containing a portion of cytoplasm within its lumen. The autophagosome then undergoes maturation by fusing with lysosomes to form an autophagolysosome. This process is again mediated by Beclin-1 as well as LC3 on the autophagosome. Following maturation, the captured cytoplasmic contents are degraded by lysosomal proteases for reuse by the cell.
Autophagy typically occurs continuously at a low level but is increased in response to cellular stresses such as nutrient deprivation in order to provide a supply of nutrients to aid cell survival. In addition to this survival function, autophagy plays other important roles that are the focus of this review.
Viruses are obligate intracellular parasites and therefore totally dependent upon a host cell for replication. This dependence has led viruses to develop ways of harnessing normal cellular processes for their own purposes. A diverse range of viruses has now been shown to manipulate autophagy. Some viruses stimulate autophagy to aid their replication. Infection with poliovirus induces the formation of membranes resembling double-membrane autophagosomes that contribute to virus production (Suhy et al., 2000; Jackson et al., 2005). Rotavirus also stimulates the formation of autophagosome-like structures with the virus replicating in close proximity to these vesicles (Berkova et al., 2006). In contrast, autophagy poses a threat for other viruses and a range of viral countermeasures have been identified (Jordan and Randall, 2011). Autophagy inhibition has been well characterized for members of the herpesvirus family. In humans, eight herpesviruses have been identified that are divided into three sub-families (alpha-, beta-, and gammaherpesvirinae). These divisions were historically based on cellular tropism but are now based on viral genome organization (Table 1). Viruses across all sub-families are able to manipulate autophagy, employing a range of different mechanisms. These mechanisms and the effects on both viral replication and the host antiviral immune response are described below.
HSV-1 Inhibits Autophagy to Prevent Viral Degradation
Autophagy has been shown to play important roles in many pathways of the innate immune system. These roles include modulating pathogen recognition receptors (Lee et al., 2007; Takeshita et al., 2008), controlling the production of key innate cytokines (Lee et al., 2007; Saitoh et al., 2008), and controlling phagocytosis (Sanjuan et al., 2007; Zang et al., 2012). Autophagy can directly contribute to innate immune control of viruses within infected cells, a role identified in the first study on Beclin-1 (Liang et al., 1998). Overexpression of Beclin-1 in neurons protected mice against fatal infection with the neurotropic alphavirus Sindbis (Note: Sindbis is a member of the Togaviridae family and not a herpesvirus; Liang et al., 1998). This protection resulted from the degradation of the Sindbis capsid protein by autophagy (Orvedahl et al., 2010). A range of intracellular bacteria and viruses are now known to be degraded by autophagy, a process termed xenophagy (Rich et al., 2003; Gutierrez et al., 2004; Nakagawa et al., 2004; Talloczy et al., 2006). The alphaherpesvirus herpes simplex virus type 1 (HSV-1) provided the first evidence of a pathogen inhibiting autophagy to escape xenophagy. Within virus-infected cells activation of cellular dsRNA-dependent protein kinase R (PKR) inhibits host and viral protein translation and viral replication. HSV-1 encodes a protein, ICP34.5, which inhibits this PKR-dependent antiviral activity (Chou et al., 1990). Talloczy et al. (2002) demonstrated that ICP34.5 also prevents PKR-dependent activation of autophagy in HSV-1-infected cells. Subsequent research revealed that ICP34.5 binds the essential autophagy gene Beclin-1 preventing the formation of autophagosomes (Orvedahl et al., 2007). Deletion of the Beclin-1 binding domain from ICP34.5 did not alter viral growth in vitro but could decrease viral growth and neurovirulence in a mouse model. Note that this decrease in neurovirulence could be due to a number of different factors. For example, survival of infected cells might be increased by the presence of a functional autophagy pathway. However, two observations support a contribution from xenophagy in reducing HSV-1 neurovirulence. First, HSV-1 virions can be observed in vesicles that morphologically resemble autophagosomes (Smith and de Harven, 1978; Talloczy et al., 2006). Second, biochemical studies show that inhibiting autophagy decreases the rate of degradation of HSV-1 proteins (Talloczy et al., 2006).
Herpesviruses Differ in their Ability to Inhibit Autophagy
Other herpesviruses have also been shown to possess autophagy inhibitory mechanisms. In cells infected with the betaherpesvirus human cytomegalovirus (HCMV) autophagy is initially activated before being inhibited 24 h post infection (Chaumorcel et al., 2008; McFarlane et al., 2011). HCMV-mediated autophagy inhibition requires de novo viral protein synthesis and is mediated by the viral protein TRS1 (Chaumorcel et al., 2012). Interestingly, TRS1 appears to be a functional homolog of the HSV protein ICP34.5, interacting with both PKR and Beclin-1. Like ICP34.5 it is the interaction between TRS1 and Beclin-1 that inhibits autophagy.
The human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) also inhibits autophagy by targeting Beclin-1 but uses a different mechanism to do so. The cellular Bcl-2 protein inhibits apoptosis but can also inhibit autophagy by binding Beclin-1(Pattingre et al., 2005). KSHV encodes a viral homolog of Bcl-2, called orf16, that can similarly bind Beclin-1 and inhibit autophagy (Pattingre et al., 2005). It is not currently known whether the two viral Bcl-2 homologs (BHRF1 and BALF1) encoded by the other human gammaherpesvirus Epstein–Barr virus (EBV) can similarly inhibit autophagy. However, it seems likely that one or both of these viral proteins will do so given that the viral Bcl-2 homolog encoded by another gammaherpesvirus, in this case murine MHV68, inhibits autophagy (Pattingre et al., 2005). Strong evidence for the importance of autophagy inhibition by these viral Bcl-2 homologs is provided by the MHV68 system. Mutating the Beclin binding domain of the MHV68 Bcl-2 homolog did not effect the establishment of viral latency but impaired the ability of the virus to maintain chronic infection in mice (E et al., 2009).
Interestingly, the protein interacting with carboxy terminal 1 (PICT-1), a putative human tumor suppressor protein of unknown function, has recently been shown to bind KSHV Bcl-2 (Kalt et al., 2011). Ectopic expression of PICT-1, which contains two nuclear localization sequences (NLS) and a nucleolar localization signal, caused KSHV Bcl-2 to relocalize from its normal mitochondrial localization into nucleoli thereby inhibiting its anti-apoptotic activity. The ability of PICT-1 to counteract KSHV Bcl-2-mediated autophagy inhibition was not examined in the paper. However, since Beclin-1 is almost entirely located in the cytoplasm and acts in this compartment (Liang et al., 2001), sequestration in the nucleus may well interfere with the ability of KSHV Bcl-2 to inhibit autophagy. If this proves to be the case then PICT-1 may represent the first example of a cellular countermeasure against viral inhibition of autophagy.
All of the herpesvirus autophagy inhibitory proteins described thus far act by interacting with Beclin-1. KSHV has developed an additional inhibitory mechanism, targeting a separate component of the cellular autophagy machinery. The viral FLICE like inhibitor protein (v-FLIP), a homolog of a cellular (c-FLIP), can inhibit both apoptosis and autophagy (Lee et al., 2009). v-FLIP binds Atg3 preventing it from processing LC3 thereby inhibiting autophagosome formation. Overexpression of either c-FLIP or v-FLIP inhibited cell death induced by rapamycin stimulation of autophagy (Lee et al., 2009).
In contrast to the herpesviruses described above varicella-zoster virus (VZV) is unable to inhibit autophagy. Indeed, autophagy is rapidly activated following VZV infection with many autophagosomes present in infected cells in vitro and in infected cells within skin vesicles (Takahashi et al., 2009; Carpenter et al., 2011). Unlike the other neurotropic alpha herpesviruses HSV-1 and HSV-2, VZV lacks an ICP34.5 ortholog; VZV also lacks a viral Bcl-2 homolog. The high level of autophagy present in VZV-infected cells may be the cause of the low titers of this virus obtained in vitro. It is interesting to note that an inability to inhibit autophagy has not proved detrimental to VZV; almost everyone is infected with the virus and in that regard it is a highly successful pathogen. Indeed, it has been postulated that VZV-induced autophagy may be beneficial to the virus, prolonging the survival of infected cells by reducing endoplasmic reticulum stress (Carpenter et al., 2011).
Autophagy and the Adaptive Immune Response to Herpesviruses
Herpesviruses have evolved an arsenal of immune evasion mechanisms in order to establish life-long latent infection of immunocompetent hosts (Horst et al., 2011). As well as playing a key role in innate anti-viral immunity, autophagy is also important in adaptive immunity and autophagy manipulation by herpesviruses affects this arm of the immune response. In agreement with the earlier work suggesting xenophagy could reduce HSV-1 neurovirulence, strains of HSV-1 incapable of inhibiting autophagy (due to deletion of the Beclin-1 binding domain of ICP34.5) caused less disease following corneal infection of mice (Leib et al., 2009). However, no disease decrease was observed for Rag1 knockout mice that lack B and T cells suggesting that the protective effect was mediated by adaptive, rather than innate, immunity. In accordance with this result mice infected with the mutant virus had higher virus-specific CD4+ T cell responses compared to animals infected with wild-type virus. While autophagy can reduce HSV-1-mediated disease the relative contributions made by the innate and adaptive immune responses are currently unclear and may differ depending upon the model system being used.
Viral inhibition of autophagy can potentially limit adaptive immunity by acting at two key stages. First, autophagy within virus-infected cells may be important for the generation of cross-primed CD8+ T cell responses by dendritic cells (DCs). Inhibiting autophagy in influenza-infected cells reduced their ability to stimulate influenza-specific CD8+ T cells in mice (Uhl et al., 2009). Similar results have also been observed using a tumor antigen model (Li et al., 2008). Second, autophagy provides a route for antigens to enter antigen-processing pathways within pathogen-infected cells, allowing them to be processed and presented to antigen-specific T cells. Since the first landmark observation that autophagy can generate an MHC II epitope from a self antigen (Brazil et al., 1997) several pathogen-encoded antigens have been shown to be processed this way. These antigens include the bacterial protein neomycin phosphotransferase (NeoR; Nimmerjahn et al., 2003), the mycobacterial antigen Ag85B (Jagannath et al., 2009), and EBV nuclear antigen 1 (EBNA1), the first example of a viral protein processed by autophagy for recognition by CD4+ T cells (Paludan et al., 2005). Autophagy can also generate MHC class I epitopes, the first example being arising from studies on HSV-1 infection of murine macrophages (English et al., 2009). As expected, autophagy was inhibited in the HSV-1-infected cells by ICP34.5. These infected cells also presented an MHC class I restricted viral epitope generated using the classical proteasome-dependent pathway. However, at later stages of infection higher levels of the epitope were generated and an additional processing pathway was involved that was sensitive to bafilomycin A and 3-methyladenine, inhibitors of the vacuolar processing pathway and autophagy respectively. In addition to normal double membrane autophagosomes, the HSV-infected cells also contained unusual four layered membrane structures originating from the nucleus that, based on the presence of LC3, appear to be autophagosomes. These unusual nuclear-derived autophagosomes were not observed in cells treated with rapamycin to stimulate autophagy nor cells infected with recombinant HSV-1 lacking ICP34.5. They may therefore represent a cellular response to a combination of HSV-1 infection and ICP34.5-mediated blockade of conventional autophagy. A second interesting observation from this work was that presentation of the viral MHC class I epitope by cells infected with HSV-1 lacking ICP34.5 was also sensitive to bafilomycin A and therefore involved the vacuolar pathway. As these cells lacked the four layered autophagosomes this result may indicate that classical double membrane autophagosomes also contribute to the generation of MHC class I epitopes.
Nuclear Shelter – Another Strategy to Evade Autophagy
Upon establishing latent infection, the herpesvirus genome persists as an episome within the nucleus. To ensure the viral genome is replicated and segregated into daughter cells the gammaherpesviruses EBV and KSHV each express a genome maintenance protein (EBNA1 and latency-associated nuclear antigen (LANA) respectively). Deletion of EBNA1 decreased EBV growth transformation 10,000-fold, clearly demonstrating the vital role of such proteins (Humme et al., 2003). Although loss of EBNA1 can be compensated by integration of the EBV genome into cellular DNA, this is a very rare event, again highlighting the importance of a correctly functioning genome maintenance protein for the gammaherpesviruses.
The dependence on the continuous presence of genome maintenance proteins to ensure life-long viral persistence represents a potential Achiles' heel for these viruses. Logically, one would therefore expect genome maintenance proteins to be well protected from immunological surveillance. This is indeed the case. EBNA1 contains a large internal glycine/alanine repeat (GAr) domain that decreases EBNA1 processing and presentation by the HLA class I processing pathway via several complementary mechanisms (Levitskaya et al., 1997; Tellam et al., 2008; Apcher et al., 2009). The KSHV genome maintenance protein LANA also contains an internal repeat domain, in this case consisting of acidic residues, that similarly reduces this protein's synthesis (Kwun et al., 2007) and degradation (Zaldumbide et al., 2007). In contrast to short-lived proteins, which are generally thought to be degraded by the proteasome, long-lived proteins are thought to be degraded by autophagy; measuring the degradation of long-lived proteins is a classical method for measuring the autophagy activity of cells (Klionsky et al., 2008). The genome maintenance proteins might therefore be potential substrates for autophagic degradation. This is indeed the case for EBNA1, which was the first viral protein identified as being degraded by autophagy (Paludan et al., 2005). Furthermore, two different EBNA1-specific CD4+ T cell clones were able to recognize EBV-transformed B lymphoblastoid cell lines (LCLs) in an autophagy dependant manner, demonstrating that such degradation could expose EBNA1 to the immune system. Remarkably, of all the EBV latent cycle proteins EBNA1 contains the greatest number of MHC class II epitopes and most individuals possess strong CD4+ T cell immunity against this protein (Munz et al., 2000; Leen et al., 2001; Hislop et al., 2007). Taken together, these observations appear paradoxical: how can EBV persist in MHC class II positive B cells despite strong CD4+ T cell responses specific for EBNA1, the protein critical for viral persistence?
In contrast to the observations described above, other research groups using CD4+ T cell clones specific for other EBNA1 epitopes found these cells were unable to recognize LCLs (Khanna et al., 1997; Mautner et al., 2004). In our laboratory, CD4+ T cell clones specific for two epitopes could recognize LCLs expressing natural levels of the EBNA1 protein while clones specific for a third epitope could not (Leung et al., 2010). The discordant results reported by different laboratories may therefore reflect real differences in the presentation of different epitopes from this protein. Although cell surface levels were low, control experiments confirmed that the two epitopes presented by LCLs were indeed endogenously accessing the MHC II pathway. Interestingly, only one epitope was autophagy-dependent, the other being processed by another intracellular route that is currently under investigation. Different epitopes from the same source protein being processed by different routes has been observed previously for influenza hemagglutinin (Sinnathamby and Eisenlohr, 2003). When EBNA1 was over-expressed as a nuclear protein, surface levels of the two naturally presented epitopes increased slightly, but the processing routes remained the same. However, expressing EBNA1 as a cytoplasmic protein, through mutation of its NLS, resulted in all three epitopes being processed by autophagy and presented at high levels by cells. This striking result was a direct result of EBNA1's new cytoplasmic localization since addition of a heterologous NLS reversed the effect (Leung et al., 2010). While EBNA1 needs to be localized to the nucleus for its correct functioning in the virus life cycle, nuclear localization clearly brings an additional benefit, namely limiting EBNA1's endogenous processing by macroautophagy and presentation to CD4+ T cells. Potentially "nuclear shelter" (Leung and Taylor, 2010) could also help reduce CD8+ T cell presentation of EBNA1. Reducing protein turnover may decrease the need for newly synthesized replacement polypeptides that are the dominant source of MHC class 1 peptides from EBNA1 (Lee et al., 2004; Tellam et al., 2004; Voo et al., 2004; Mackay et al., 2009).
Are other nuclear-localized proteins sheltered from macroautophagy like EBNA1? Studies using non-viral proteins suggest this may be the case. The huntingtin and ataxin-1 proteins each contain a large poly-glutamine repeat that can cause these proteins to aggregate in cells. Aggregates of huntingtin or ataxin-1 were degraded by autophagy in the cytoplasm but not in the nucleus (Iwata et al., 2005). A mutation in the NLS of a tumor antigen causing it to relocalize out of the nucleus enhanced its CD4+ T cell recognition (Wang et al., 1999). Although it is not known if this epitope was processed by autophagy, the observation is consistent with the EBNA1 data. In contrast to these examples, presentation of an autophagy-dependant epitope from the NeoR protein was not reduced by the addition of a heterologous NLS (Riedel et al., 2008). The reason for this different result is unclear, but one possibility is that it might reflect different rates of nuclear import. Previous work has shown that although the addition of a minimal NLS allows nuclear import, this occurs with slow kinetics unless additional flanking sequences are included (Rihs and Peters, 1989). The import rate of the NLS-containing NeoR protein has not been measured, however EBNA1 is rapidly imported following its translation suggesting that levels of cytoplasmic EBNA1 are likely to be very low (Kitamura et al., 2006). Finally, it is important to note that other endogenous MHC class II processing pathways exist. Some of these pathways involve the proteasome (Delmas et al., 2005; Tewari et al., 2005), others CMA (Zhou et al., 2005) while others have not been mechanistically defined. Antigen localization can affect processing by one of these alternative pathways. The CMV nuclear protein IE1 contains an MHC class II epitope that is not processed by autophagy but by a proteasome-dependent mechanism. Although relocalization of IE1 into the cytoplasm did not alter the degree of presentation of this epitope, the mechanism of presentation was altered (Delmas et al., 2005).
How do Nuclear-Localized Antigens Enter the Autophagy Pathway?
The fact that some EBNA1 MHC class II epitopes are autophagy-dependent raises the interesting question of where in the cell does the nuclear-localized protein access the autophagic pathway? Can autophagy occur in the nucleus? The best-studied example of "nucleophagy" is Piecemeal Microautophagy of the Nucleus (PMN), a form of microautophagy that occurs in yeast (Roberts et al., 2003). During PMN, small pieces of the nucleus are pinched off into the yeast degradative vacuole; PMN is therefore a relatively gentle and selective process that can be tolerated by the cell. A much more dramatic example of nucleophagy has been observed in the filamentous fungus Aspergillus oryzae. Here, entire nuclei are degraded after being surrounded by large (1–2 μm diameter) autophagosomes (Shoji et al., 2010). While such drastic loss of genetic material would be lethal for any mononuclear cell, it can be tolerated within the multi-nucleated hyphae of this organism. Since autophagy is important for the growth of this fungus under starvation conditions, nucleophagy may be acting to recycle the constituents of redundant nuclei for use at growing hyphal tips.
Do mammalian cells undergo nucleophagy? The extent to which PMN takes place in other species is unknown, but homologs of the yeast genes essential for PMN, Vac8, and Nvj1, have not been identified in animal cells (Mijaljica et al., 2010). Granting non-specific macroautophagy access to the nucleus could result in irreversible loss of genetic material with catastrophic consequences for the cell. Indeed, to guard against such loss mammalian cells inhibit autophagy during mitosis, a time when chromosomes enter the cytoplasm following breakdown of the nuclear membrane (Eskelinen et al., 2002). However, nucleophagy has been observed in mammalian cells in select circumstances. In mouse macrophages HSV-1 infection triggers an unusual form of autophagy involving four-layer autophagosomes emerging from the nuclear envelope (English et al., 2009). It is not known whether these unusual autophagosomes can form in human cells infected with HSV-1, or if other herpesviruses can trigger their formation.
Other examples of nucleophagy occur in the context of genetic mutations. In Bloom's syndrome, a rare cancer-predisposing disorder caused by mutation of the BLM gene, micronuclei bud from the nucleus into the cytoplasm (Yankiwski et al., 2000). The extent to which these cytoplasmic micronuclei are substrates of autophagy and the ability of normal cells to release micronuclei are currently not known. Finally, cells with fragile nuclei, caused by mutations in genes encoding nuclear lamina proteins, have been observed to contain perinuclear autophagosomes/autolysosomes-containing nuclear components (Park et al., 2009). Intriguingly, the authors report similar autophagosomes/autolysosomes in wild-type cells, although at a much lower frequency.
Autophagy is now known to exhibit greater selectivity for its substrates than once thought. Several autophagy adaptor proteins have now been shown to transport a range of substrates to autophagosomes for degradation. These substrates include aggregated or ubiquitinated proteins, damaged mitochondria, and intracellular bacteria (reviewed in Johansen and Lamark, 2011). Selective autophagy could be another route for nuclear proteins to enter cytoplasmic autophagosomes, one that could operate without causing genetic loss. One of the autophagy adaptor proteins, p62 (also called SQSTM1), has recently been shown to deliver capsids of Sindbis virus to autophagosomes (Orvedahl et al., 2010). With respect to the selective autophagy of nuclear proteins, p62 is of particular interest since it contains both nuclear localization and nuclear export sequences and consequently shuttles rapidly between the nucleus and cytoplasm (Pankiv et al., 2010). However, it is not yet known whether p62 is involved in the selective degradation of nuclear proteins by autophagy.
Activation of Autophagy by Herpesviruses
Thus far we have considered autophagy as a threat to herpesviruses. However, the relationship between herpesviruses and autophagy is more complex and several examples exist of herpesviruses activating autophagy at different stages in their lifecycle. Autophagy activation can occur during lytic infection. As described earlier autophagy is rapidly activated in VZV-infected cells because the virus apparently lacks mechanisms to inhibit this pathway. However, autophagy activation has also been reported for viruses that do possess such mechanisms, suggesting that autophagy may be beneficial for viruses at particular stages of their replicative cycle. Although HSV-1 encodes the autophagy inhibitor ICP34.5 this virus has been reported to rapidly activate autophagy in human fetal foreskin fibroblasts (McFarlane et al., 2011). Autophagy induction by HSV-1 has also been reported in murine macrophages, although in this case classical macroautophagy still appeared to be inhibited by virus-encoded ICP34.5 and a morphologically distinct type of macroautophagy was observed (English et al., 2009). CMV infection also rapidly activates autophagy that is subsequently inhibited by the CMV-encoded protein TRS1 (McFarlane et al., 2011; Chaumorcel et al., 2012). KSHV also appears to activate autophagy during lytic reactivation. The KSHV protein RTA is able to activate autophagy, inhibiting this process reduced RTA-mediated lytic gene expression and KSHV replication suggesting autophagy plays a key role in the replication of KSHV during lytic cycle (Wen et al., 2010). Autophagy activation can also occur during herpesvirus latency. The EBV-encoded latent membrane protein 1 (LMP1) induces autophagy in EBV transformed B cells (Lee and Sugden, 2008). This induction does not result from LMP1's well-documented activation of cellular signaling pathways but from the presence of three of the molecule's transmembrane spanning domains. Since LMP1 is also degraded by autophagy these domains, which had previously not been assigned a functional role, may provide LMP1 with a feedback mechanism that regulates cellular levels of the protein.
During their evolution, the herpesviruses have developed a range of mechanisms to interact with the host autophagy machinery. Under some circumstances these viruses need to evade host immunity and this is achieved in several ways. One way is to target key cellular proteins required for autophagy. In this regard, several viruses have independently evolved mechanisms that target Beclin-1, reflecting the key role this protein plays in autophagy. The targeting of another autophagy protein, ATG3, by KSHV raises the possibility that a range of inhibitory mechanisms exist, waiting to be discovered. Another way to evade immunity may be through localization of genome maintenance proteins into the nucleus, thereby minimizing their access to the autophagy machinery. In this review, we have focused on macroautophagy, which is the best characterized of the three currently defined autophagy pathways. The other pathways are beginning to be elucidated and one of them, CMA, has been shown to generate an MHC class II restricted epitope from a non-viral antigen (Zhou et al., 2005). However, the contribution of these other pathways to viral immunity, and whether they too are inhibited by herpesviruses, is currently unknown. Finally, a complex balance exists between herpesviruses and autophagy within the host cell and under some circumstances herpesviruses activate autophagy, presumably for their benefit. Two recent observations – PICT-1-mediated relocalization of KSHV Bcl-2 away from its site of action, and nuclear-derived autophagosomes in mouse macrophages infected with HSV-1 – raise the possibility that cells possess mechanisms to counteract viral manipulation of autophagy. Understanding the balance between autophagy inhibition and activation within virally infected cells, and the consequences for anti-viral immunity, presents a challenge and an opportunity for future research.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Klionsky, D. J., Abeliovich, H., Agostinis, P., Agrawal, D. K., Aliev, G., Askew, D. S., Baba, M., Baehrecke, E. H., Bahr, B. A., Ballabio, A., Bamber, B. A., Bassham, D. C., Bergamini, E., Bi, X., Biard-Piechaczyk, M., Blum, J. S., Bredesen, D. E., Brodsky, J. L., Brumell, J. H., Brunk, U. T., Bursch, W., Camougrand, N., Cebollero, E., Cecconi, F., Chen, Y., Chin, L. S., Choi, A., Chu, C. T., Chung, J., Clarke, P. G., Clark, R. S., Clarke, S. G., Clave, C., Cleveland, J. L., Codogno, P., Colombo, M. I., Coto-Montes, A., Cregg, J. M., Cuervo, A. M., Debnath, J., Demarchi, F., Dennis, P. B., Dennis, P. A., Deretic, V., Devenish, R. J., Di Sano, F., Dice, J. F., Difiglia, M., Dinesh-Kumar, S., Distelhorst, C. W., Djavaheri-Mergny, M., Dorsey, F. C., Droge, W., Dron, M., Dunn, W. A. Jr., Duszenko, M., Eissa, N. T., Elazar, Z., Esclatine, A., Eskelinen, E. L., Fesus, L., Finley, K. D., Fuentes, J. M., Fueyo, J., Fujisaki, K., Galliot, B., Gao, F. B., Gewirtz, D. A., Gibson, S. B., Gohla, A., Goldberg, A. L., Gonzalez, R., Gonzalez-Estevez, C., Gorski, S., Gottlieb, R. A., Haussinger, D., He, Y. W., Heidenreich, K., Hill, J. A., Hoyer-Hansen, M., Hu, X., Huang, W. P., Iwasaki, A., Jaattela, M., Jackson, W. T., Jiang, X., Jin, S., Johansen, T., Jung, J. U., Kadowaki, M., Kang, C., Kelekar, A., Kessel, D. H., Kiel, J. A., Kim, H. P., Kimchi, A., Kinsella, T. J., Kiselyov, K., Kitamoto, K., Knecht, E., Komatsu, M., Kominami, E., Kondo, S., Kovács, A. L., Kroemer, G., Kuan, C. Y., Kumar, R., Kundu, M., Landry, J., Laporte, M., Le, W., Lei, H. Y., Lenardo, M. J., Levine, B., Lieberman, A., Lim, K. L., Lin, F. C., Liou, W., Liu, L. F., Lopez-Berestein, G., López-Otín, C., Lu, B., Macleod, K. F., Malorni, W., Martinet, W., Matsuoka, K., Mautner, J., Meijer, A. J., Meléndez, A., Michels, P., Miotto, G., Mistiaen, W. P., Mizushima, N., Mograbi, B., Monastyrska, I., Moore, M. N., Moreira, P. I., Moriyasu, Y., Motyl, T., Münz, C., Murphy, L. O., Naqvi, N. I., Neufeld, T. P., Nishino, I., Nixon, R. A., Noda, T., Nürnberg, B., Ogawa, M., Oleinick, N. L., Olsen, L. J., Ozpolat, B., Paglin, S., Palmer, G. E., Papassideri, I., Parkes, M., Perlmutter, D. H., Perry, G., Piacentini, M., Pinkas-Kramarski, R., Prescott, M., Proikas-Cezanne, T., Raben, N., Rami, A., Reggiori, F., Rohrer, B., Rubinsztein, D. C., Ryan, K. M., Sadoshima, J., Sakagami, H., Sakai, Y., Sandri, M., Sasakawa, C., Sass, M., Schneider, C., Seglen, P. O., Seleverstov, O., Settleman, J., Shacka, J. J., Shapiro, I. M., Sibirny, A., Silva-Zacarin, E. C., Simon, H. U., Simone, C., Simonsen, A., Smith, M. A., Spanel-Borowski, K., Srinivas, V., Steeves, M., Stenmark, H., Stromhaug, P. E., Subauste, C. S., Sugimoto, S., Sulzer, D., Suzuki, T., Swanson, M. S., Tabas, I., Takeshita, F., Talbot, N. J., Tallóczy, Z., Tanaka, K., Tanaka, K., Tanida, I., Taylor, G. S., Taylor, J. P., Terman, A., Tettamanti, G., Thompson, C. B., Thumm, M., Tolkovsky, A. M., Tooze, S. A., Truant, R., Tumanovska, L. V., Uchiyama, Y., Ueno, T., Uzcátegui, N. L., van der Klei, I., Vaquero, E. C., Vellai, T., Vogel, M. W., Wang, H. G., Webster, P., Wiley, J. W., Xi, Z., Xiao, G., Yahalom, J., Yang, J. M., Yap, G., Yin, X. M., Yoshimori, T., Yu, L., Yue, Z., Yuzaki, M., Zabirnyk, O., Zheng, X., Zhu, X., and Deter, R. L. (2008). Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4, 151–175.
Kwun, H. J., Da Silva, S. R., Shah, I. M., Blake, N., Moore, P. S., and Chang, Y. (2007). Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 mimics Epstein–Barr virus EBNA1 immune evasion through central repeat domain effects on protein processing. J. Virol. 81, 8225–8235.
Lee, D. Y., and Sugden, B. (2008). The latent membrane protein 1 oncogene modifies B-cell physiology by regulating autophagy. Oncogene 27, 2833–2842.
Lee, H. K., Lund, J. M., Ramanathan, B., Mizushima, N., and Iwasaki, A. (2007). Autophagy-dependent viral recognition by plasmacytoid dendritic cells. Science 315, 1398–1401.
Lee, J. S., Li, Q., Lee, J. Y., Lee, S. H., Jeong, J. H., Lee, H. R., Chang, H., Zhou, F. C., Gao, S. J., Liang, C., and Jung, J. U. (2009). FLIP-mediated autophagy regulation in cell death control. Nat. Cell Biol. 11, 1355–1362.
Lee, S. P., Brooks, J. M., Al-Jarrah, H., Thomas, W. A., Haigh, T. A., Taylor, G. S., Humme, S., Schepers, A., Hammerschmidt, W., Yates, J. L., Rickinson, A. B., and Blake, N. W. (2004). CD8 T cell recognition of endogenously expressed Epstein–Barr virus nuclear antigen 1. J. Exp. Med. 199, 1409–20.
Leen, A., Meij, P., Redchenko, I., Middeldorp, J., Bloemena, E., Rickinson, A., and Blake, N. (2001). Differential immunogenicity of Epstein–Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses. J. Virol. 75, 8649–8659.
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Leung, C. S., Haigh, T. A., Mackay, L. K., Rickinson, A. B., and Taylor, G. S. (2010). Nuclear location of an endogenously expressed antigen, EBNA1, restricts access to macroautophagy and the range of CD4 epitope display. Proc. Natl. Acad. Sci. U.S.A. 107, 2165–2170.
Leung, C. S., and Taylor, G. S. (2010). Nuclear shelter: The influence of subcellular location on the processing of antigens by macroautophagy. Autophagy 6, 560–561.
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Orvedahl, A., Macpherson, S., Sumpter, R. Jr., Talloczy, Z., Zou, Z., and Levine, B. (2010). Autophagy protects against Sindbis virus infection of the central nervous system. Cell Host Microbe 7, 115–127.
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Pankiv, S., Lamark, T., Bruun, J. A., Overvatn, A., Bjorkoy, G., and Johansen, T. (2010). Nucleocytoplasmic shuttling of p62/SQSTM1 and its role in recruitment of nuclear polyubiquitinated proteins to promyelocytic leukemia bodies. J. Biol. Chem. 285, 5941–5953.
Park, Y. E., Hayashi, Y. K., Bonne, G., Arimura, T., Noguchi, S., Nonaka, I., and Nishino, I. (2009). Autophagic degradation of nuclear components in mammalian cells. Autophagy 5, 795–804.
Pattingre, S., Tassa, A., Qu, X., Garuti, R., Liang, X. H., Mizushima, N., Packer, M., Schneider, M. D., and Levine, B. (2005). Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122, 927–939.
Rich, K. A., Burkett, C., and Webster, P. (2003). Cytoplasmic bacteria can be targets for autophagy. Cell. Microbiol. 5, 455–468.
Riedel, A., Nimmerjahn, F., Burdach, S., Behrends, U., Bornkamm, G. W., and Mautner, J. (2008). Endogenous presentation of a nuclear antigen on MHC class II by autophagy in the absence of CRM1-mediated nuclear export. Eur. J. Immunol. 38, 2090–2095.
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Roberts, P., Moshitch-Moshkovitz, S., Kvam, E., O'Toole, E., Winey, M., and Goldfarb, D. S. (2003). Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae. Mol. Biol. Cell 14, 129–141.
Sahu, R., Kaushik, S., Clement, C. C., Cannizzo, E. S., Scharf, B., Follenzi, A., Potolicchio, I., Nieves, E., Cuervo, A. M., and Santambrogio, L. (2011). Microautophagy of cytosolic proteins by late endosomes. Dev. Cell 20, 131–139.
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Keywords: antigen, CD4, EBNA1, EBV, macroautophagy, nucleophagy, presentation, processing
Citation: Williams LR and Taylor GS (2012) Autophagy and immunity – insights from human herpesviruses. Front. Immun. 3:170. doi: 10.3389/fimmu.2012.00170
Received: 24 January 2012; Paper pending published: 20 February 2012;
Accepted: 05 June 2012; Published online: 04 July 2012.
Edited by:Christian Munz, University of Zurich, Switzerland
Reviewed by:Philippe Pierre, Centre National de la Recherche Scientifique, France
Matthew Albert, Institut Pasteur, France
Copyright: © 2012 Williams and Taylor. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.
*Correspondence: Graham S. Taylor, School of Cancer Sciences, University of Birmingham, Vincent Drive, Birmingham, B15 2TT UK. e-mail: firstname.lastname@example.com


text:
It eats you away inside out
It nibbles away at your pleasure
it feasts away on your dreams
It laughs at your issues
It toys with your emotion
It plays with your heart.
Your hearts raises
Your hand tremble
Your stomach drops
There is nothing you can do
But pluck your hair out of your head
Because the silent killer is here
Waiting to take you to the door.
©2018. VishalDutia. All Rights Reversed.

subset: commoncrawl


text:
Fayette County
July 17 proclaimed 'Jackie French Day' by mayor
Lexington-Fayette Urban County Government employee Jackie French was honored recently for 38 years of service at Parks and Recreation.
During the July 17 Super Sunday basketball showcase, Council Member James Brown presented French with a proclamation from Mayor Jim Gray declaring that day as "Jackie French Day" in Lexington.
Super Sunday is annual basketball event, organized in part by French, who has also organized the Lexington-Fayette Urban County Government men's adult "Dirt Bowl" basketball league since 1979.
During the event, French also received a Certificate of Appreciation plaque from the Georgetown Street Area Neighborhood Association for her service and contributions toward the sustainable development of Douglass Park.
For nearly 40 years, Super Sunday has been held at Douglass Park and features Lexington-Fayette Urban County Government men's summer league games throughout the day.


text:
So sweet. So good.
And so off limits.
But I'm about to push those limits.
And I won't stop 'til she cries uncle.
Mira. Body of a goddess.
Pure perfection.
She walks in and I know I'll make her mine.
She likes what she sees.
Wants a piece of me.
And I'm about to give it to her.
I don't care if she's my brother's new step-daughter.
I don't care what s#*t goes down.
Or the fact that her company is on the line.
Not anymore.
Because he only thing going down now…
Will be one dirty uncle.


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text:
- Much quieter week, thank goodness. Just spent some time working on
what awards I might be eligible to win for my book and preparing
applications where necessary. And mostly reading and catching up on
personal projects, plus doing "responsible" stuff like paying bills. I
REALLY need to answer e-mail though.
- I try to do a video
for each of my two channels once a month, and one of my webcomics
updates every week while the other updates every month. With all the
excitement and attention of September, it was hard for me to meet those
goals, but I did manage to get one more video and the monthly webcomic
done in September, so I'm happy that I still met my self-imposed goals.
- I beat up on my Pitch Wars manuscripts a little and did some revising on my science fiction romance novel.
- Got my September newsletter sent out. (You can sign up to receive the newsletter here. It doesn't come out often--just when I have news, so only six times since I started it around a year ago.)
- Completed some interview questions for a UK magazine and set up some stuff to be interviewed on the BBC again.
- I ate at IHOP with Jeaux this week. Then we went to my place and chatted and listened to the newest Night Vale episode.
- A.S.P.I.E. - Autism Spectrum Pride In Everything Facebook group linked my Salon article and discussed its applicability.
- YouthVoices.net mentioned me.
- cassandraswarning on Blogger is discussing asexuality and mentions my book.
- There was also a gross writeup on a "political" site (I say "political" with scare quotes on purpose). I mentioned it in a previous blog post. It mentioned my book as it mocked asexuality, but I really don't think I need to link to something that was constructed specifically to be dismissive and vicious, not constructive or respectfully critical. It was just a hate-wank fest that deliberately redefined our terminology so they could mock it and laughed uproariously at the idea that we need to have any organizations or communities because we're literally all about "not doing anything."
- Finished reading: The Mother Tongue: English and How It Got That Way by Bill Bryson. Four-star review.
- Currently reading: Spider's Bite by Jennifer Estep.
New singing performances:
Here I'm singing Limahl's "Never Ending Story." Nostalgic and fun to sing, especially with these harmonies.
New drawings:
Webcomic Negative One Issue 0490: "The End of Wanting."
Webcomic So You Write Issue 40: "The Big Lie."
New videos:
Here's a short one about advice authors give to each other and how important it is to find what works for you instead of slavishly committing to what works for others: Writing Every Day?
New photos:
Social media counts:
YouTube subscribers: 3,881 for swankivy (26 new this week), 383 for JulieSondra (1 new). Twitter followers: 559 for swankivy (6 new), 734 for JulieSondra (no change). Facebook: 272 friends (1 new) and 142 followers (no change) for swankivy, 473 likes for JulieSondra (23 new), 48 likes for Negative One (no change), 84 likes for So You Write (no change). Tumblr followers: 1,578 (11 new).


text:
the story in 7 dragoncons
this is the plot of a quantum fiction novel partly set in the dragoncon community. if you're here searching for dragoncon stuff, maybe you'll stay and comment on my work.
i separated the story into a series of 7s, and one of them is the cycle of years attending dragoncon in atlanta, the finest gathering of minds in the universe and the golden kernel of the new quantum age.
chapter 2, dragoncon 1
the first dragoncon. they've been going for years. neither of them could go for the last couple of years, as the job, etc has gotten more onerous. but now the boy is unemployed and living in his parents' basement, and the girl just decided she didn't want to be retail manager and stopped giving 110%. so they're at dragoncon, and it's been awhile, and they're excited to see so many different things, like how big steampunk has gotten, and so many familiar things, like the artshow stars. their friends kurt and fairy are there, they know snake vaguely. the girl attends the costuming and artshow tracks, the boy does armory and gaming, they get a tshirt each and lust after the big ticket items. the weekend will break their budget for the rest of the year, so the t-shirts will be saved for xmas. [they try them on once, take pictures. then later she gives them away, and gives the pictures for xmas, thinking since they were on their bodies once, that is good enough in quantum thinking because you can always go back to that time. he is pissed because he wanted the tshirt now and until it was stained and torn. and she'd promised a quilt of old tshirts one of these days. but she's a serious quantum-moment head four months after their vision, and doesn't want to know about heirlooms anymore.]
chapter 8, dragoncon 2
things are going along great, the game's almost finished, just needs beta testing. interest has grown remarkably, and they have a table in dc-mmo. at dragoncon they're swamped with requests from would-be beta testers, among which are spies. the alpha testers let everybody know, and hordes line up to be beta testers. the buzz about the game is enormous, and gamers pressure them to go live. the boy talks to corporate sponsors, the girl gives workshops on the new quantum age, quantum panels appear in several tracks – costuming, silk road (dance), armory (martial art), artshow and market (dc3), science, paranormal, skeptic, eff, game programming, sff lit (dc3 ). kurt does a lot of drugs to clear his head of nonquantum space, and is found at the bottom of the covered hotel pool.
chapter 15, dragoncon 3
so many tracks, so much interest. famous physics speakers, consciousness speakers, new music, new slang, new clothing styles (nano), healing, all coming together in quantum-culture [quantum aesthetics, quantum art, quantum literature, quantum politics, quantum anthropology.] there is a qAnt track now. merchandising begins. the boy and girl get offers; he wants to be rich, she is against it. there is real pressure to go corporate, with tax and accounting and postproduction and licensing, localization, advertising. snake and fairy are for it, he wants to get rich, she wants to make everybody happy.
chapter 23, dragoncon 4
the boy is important and arrogant. he's moved out of his parents' house (and out of the girl's apartment) into a trendy loft and hangs with strippers and singers and drug dealers and hiphop artists and gaming industry legends. he pretends to be running the game, but only sticks around to lead the battles, and leaves all the maintenance work to the girl, who is struggling to keep everything together in her personal life, and is beginning to have health problems. she spends too much time as admin, trying to keep up the testing, trying to fix things that are going wrong because of snake's sabotage and the boy's negligence, trying to organize people in the game, as well as being a cheerleader for the mystical and spiritual side of the game. the game is fast becoming an online live strategy game, with alien-hunts in the mountains of ring 2, and the boy's infernal piracy along the western coasts.
they start to dictate policy, the boy changes things in the game, altering rules and missions, twisting the lessons, identifying and capturing certain types of players.
the boy and girl have a big public fight over the original intentions for the game, all the way back to the text message. she argues for spreading quantum consciousness inside and outside the game, he argues for getting rich so they can do it in the sequels. there is a split in the game.
chapter 30, dragoncon 5
dragoncon is subdued this year because the game has been banned. there are spies everywhere, cameras everywhere; there's a new security rule that says no face masks, and a lot of grumbling and outright refusal (these are stopped and harassed). track participants are suspicious and quick to throw people out of panels. groups get together secretly to practice quantum exercises and to play the game. people wear symbols and develop signs and codes based on game principles. the boy and girl make an appearance together. they are mobbed, they call for action, the atmosphere becomes defiant, players march on legislative buildings, people openly play the game despite beating and detention. the boy compares himself to robin hood and issues an open challenge. the girl is terrified and goes into hiding.
chapter 34, dragoncon 6
dragoncon comes on the heels of the boy and girl's spectacular level ending in antarctica, and people flock to dragoncon instinctively, compulsively, gathering with quiet expectation; the power of the gathered multitude dampening the repressions that so ruined the mood at last year's con. the crowd grows elated as they begin for the first time to think in quantum tandem.
the moment passes and they begin to break up and go home, and the police and army move in to arrest them all. the boy is openly captured and beaten, the girl is unnoticed, in disguise, but is swept up with the others.
chapter 40, dragoncon 7
this year there is no dragoncon. the organizers have been detained, and anybody googling or writing about it on any electronic media is also detained. the game has been destroyed, quantum everything has been made punishable by death, fundamentalist classicists are viciously celebrating.
this year there are a lot of people quietly remembering dragoncon from their cells and their parents' basements and their minimum wage slavery. because of their quantum game training, they all make small entangled thoughtform minions (that are the essence of 'i want my game back') and send them scurrying about their business. billions of minions congregate together and visualize the game. it is a weak thoughtform, born of thoughtforms. but it crackles through to the real world and sends a shock thru the game community before fading out again. and then people begin to actively play the game in their imaginations and in their sleep, hooking in thru their own thoughtform minions. quickly level ! begins to fill up. they still each meet the girl, who has made an infinite run of signed authentic prints of herself.
future dragoncons are held in quantum space, where the hotels don't fill up.
Posted on January 1, 2012, in dragoncon, game, plot. Bookmark the permalink. Leave a comment.


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Article of Sena C. and Michaela L.
Valletta, the heart of Malta
Valletta is the capital city of Malta. Valletta is the smallest capital city of Europe. Malta is an independent country. The local language is Maltese and English. Maltese is a mixture of four languages, English, French, Arabic and Italian. Malta is artistic and cultural. The tourists are very important for the country. Maltese hospitality is marvelous and all visitors are welcome.
The history of Valletta
Valletta was founded in 1566 by Jean Parisot De La Valletta. After the war with the Ottomans, the city was redesigned by the architect Francesco Lapurelli. The city is built so that it looks the same to confuse the enemy during the war.
Highlights of the city buildings: discovering the beauties of Valletta
In the 15th century saw the St. John's Co-Cathedral was founded. Working in Valletta is unique, and comparable to no other capital cities in Europe. The bright houses, narrow streets, beautiful gardens and the sea view make a walk through Valletta very romantic.
Valletta nowadays' tourist attraction
The Upper Barrakka Gardens are among the quietest places. The views of Valletta Grand Harbor and Fort Ricasoli and Fort St. Angelo are simply unforgettable! The Saluting Battery a platform on the fortress walls formerly used the Knights of Malta. It served to defend the harbor with cannons and to greet friendly ships by the sea. The Church of Our Lady of Victories is the first building in Valletta. Malta 5D is a new generation theatre where you feel the "real" emotions of the movie. In a short time, you may explore the colourful and impressive episodes of the Maltese history and culture, magnified with 3D film, moving seats, water spray, air blasts and leg ticklers.
In cooperation with Stepin GmbH · Beethovenallee 21 · 53173 Bonn
Amtsgericht Bonn · HRB 8736 – Germany


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[Skip to content]
School governors are members of governing bodies, whose purpose is to help provide the best possible education for pupils at their schools. Their main function is to raise standards, monitoring progress against the targets they set.
There is information for School Governors on the main public WBC website at this link: http://www.wokingham.gov.uk/schools-and-education/school-information-and-services/school-governors/
There is also additional information for governors who are part of the Governor Traded Service on a set of pages below this level. To access them you will need to login with your school username and password. These are usually available from the School Bursar. Please click on Login at the top of the page.
When you have logged in the additional pages that are only available to schools that have purchased the Governor Services Traded Package will appear.
More information from the Governor Services Team on 0118 974 6165.


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The Batesville High School swim teams placed second at the Madison Invitational.
The Lady Bulldogs finished with a team score of 245 points. North Harrison was first with 339. Madison (196) and Silver Creek (96) finished behind the Lady Bulldogs. The BHS men recorded a team score of 237 points. Madison won the event with 305. North Harrison (201) was third followed by Silver Creek (38).
Both teams swam well, according to coach Greg McMullen.
"This was a great meet for us. Madison, Silver Creek and North Harrison are always pushing us to improve. Overall, we swam a few season best times and really put ourselves in a great position."
"Our goal from the start of the season was to improve from where we were last year and we definitely did that today."
Batesville finished 4th (men's) and 3rd (women's) in 2017-18 and were able to accumulate enough points to finish in second place behind North Harrison (women) and Madison (men).
Event winners for Batesville High School included:
– Kate Poltrack (500)
– Sean Callahan (500)
– 200 Free Relay (Maria Lopez, Sonja Gaulin, Gwen Martin, Poltrack)
– Maria Lopez (100 Breast)


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A refreshing shower is a great way to start your day off right, and a hydrating body wash can help you to nourish your skin at the same time.
• Crisp, soothing cucumber and green tea scent revitalises your senses • Light, hydrating feel and refreshing formula that effectively nourishes skin • NutriumMoisture™ technology delivers skin natural nutrients • Nourishes deep into the surface layers of the skin* • Mild, gentle formula is kind to your skin
A refreshing shower is a great way to start your day off right, and a hydrating body wash can help you to nourish your skin at the same time with Dove Fresh Touch Body Wash, a nourishing body wash with a light, refreshing cucumber and green tea scent to give you a body wash that leaves your skin feeling fresh and hydrated.This mild body wash, with NutriumMoisture™ technology, combines gentle cleansers with skin natural nutrients to give you softer& smoother skin. And with Dove Fresh Touch Body Wash, all of that comes along with an uplifting scent to revitalize your senses and help kick-start your morning. Simply squeeze a generous amount of body wash onto your hands or a shower puff and enjoy the lather and refreshing scent before rinsing. *Within the stratum corneum
Water, Sodium Laureth Sulfate, Cocamidopropyl Betaine ,Glycerin, Sodium Cocoyl Glycinate, Perfume, Sodium Chloride, Sodium Cocoyl Isethionate, Acrylates/C10-30 Alkyl Acrylate Crosspolymer, Glycol Distearate, PPG-9, Phenoxyethanol, Stearic Acid, Coconut Acid, Sodium Hydroxide, Lauric Acid, Cocamide MEA, BHT, Sodium Benzoate, Tetrasodium EDTA, Sodium Isethionate, Citric Acid, Phosphoric Acid, Oryza Sativa(Rice) Bran Oil, Methylisothiazolinone, DMDM Hydantoin, Sodium Sulfate, CI 19140, CI 61570.
Ingredients correct at time of publishing. Always check product packaging.


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The Atlantic Flight Centre welcomes all travellers to our professional full-service FBO. Located at the Saint John Airport (CYSJ) close to the shores of the famous Bay of Fundy in Saint John, New Brunswick, we take pride in our warm hospitality and professional staff who are here to assist you with all of your aviation needs.
As the heart of aviation activity in Saint John from the days of the Fundy Flying Club to today's Saint John Flying Club, hundreds of local aviation enthusiasts have been gathering at the Atlantic Flight Centre to train as pilots or to just sit and enjoy a cup of coffee to watch and talk about aviation.
Whether you are looking for a private corporate charter or a scenic tour of the historic Uptown or crystal blue waters of the St. John River and down through the famous Reversing Falls to the magnitude of the tides of the Bay of Fundy – flying above the skies of Saint John is a visual paradise.
To provide professional excellence in aircraft maintenance by experienced, skilled and dedicated Aircraft Maintenance Engineers (AME – M1 and M2) and Technicians using the highest quality products at the best possible price.
To provide a clean, comfortable lounge and fully equipped flight planning room for visiting pilots and passengers as well as courteous and friendly concierge services as needed.
The Saint John Airport (CYSJ) serves Greater Saint John and southwestern New Brunswick with daily direct flights to Toronto, Montreal and Halifax and connecting flights to Ottawa. The Saint John Airport is about a 20-minute drive from the city centre, just 26-KM, on Loch Lomond Road, Route 111 East.
Saint John Flight Services
Halifax Weather Centre


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HIC Sign-up Process
Before we can implement your new IDX search, you will need to complete the HIC agreement. Once complete, email to: email@example.com or fax to: 888-653-6026 Also, please be aware that your board charges a direct fee of $50 setup and $75 monthly (2 months must be paid in advance).
Want to Learn More about Dakno Providing Your HIC IDX?
Interested in adding a search engine friendly IDX search to your website? Learn about HIC solutions provided by Dakno by visiting our HIC IDX page.
What are the HIC Compliance Requirements?
Hawaii Central requires that all IDX participants adhere to a specific set of rules and regulations. Below is an overview of these requirements. Don't worry, we're very familiar in compliance requirements and will help ensure your website meets these standards.
HIC Disclosures, Documents and Information
Dakno Staff IDX Documentation (internal use only)


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Thursday, February 17, 2011
Crafting the Style
5 comments:
- Jill Kemerer said...
Yes! All of the details add up to style, don't they? What the character wears, drives, and the word choices he makes. Even the town he lives in adds to the style.
- February 17, 2011 at 10:12 AM
- Laura Pauling said...
So you're the mysterious Raven Requiem who has tweeted some of my posts! Nice to find you. And yes, it all comes down to showing not telling!
- February 17, 2011 at 12:06 PM
- Michele Shaw said...
Well said, and don't we all want "style." in everything we do?
- February 17, 2011 at 12:23 PM
- PW.Creighton said...
Thanks for your comments! So true Jill and Michele, we're all out to create a unique but believable and engrossing world. Laura, great to see you found your way here and extra kudos for the comment. The site of so many discarded books was a bit inspirational and depressing.
- February 17, 2011 at 9:53 PM
- Sammy Sutton Author said...
Hello PW,
Excellent, you give a writer a lot of 'food for thought' about a component we often fail to give adequate credit. I share your views about the importance of style. Well said...Thanks for sharing!
Sammy Sutton
- February 18, 2011 at 12:35 PM


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Omega has just launched a new limited edition of 18 servings with the reference number 516.52 "First Omega Wrist Chronograph". Unlike previous Omega watches, which are brand new with a "vintage-inspired" aesthetic, the Omega Wrist Chronograph combines new and vintage elements, including a fully restored Omega 3018 caliber (name for the newly restored moves). The trains are also considered with the help of their unique reputation, since the 18-inch CHRO calibers, due to their age and their situation in each of the 18 parts of the Sets will look a bit unique.
On the first debut at an event at the Omega personal museum in Biel, Switzerland, the release of the first copy Omega Chronometer Watch Wrist chronograph showed the painstaking effort Omega made to bring all steps back to existence, in a case written by the The first wrist-worn chronograph was inspired by the logo. The one hundred and twenty-five-month-old trains need not only plenty of rework and cleaning but also alternative elements – in addition to the decoration of the route. Omega considers that, in his opinion, "each of the actions is unique" as a feature of the recovery process. Omega has certainly redesigned the moves in their prestigious Omega Atelier Tourbillon store. This is a special place of White dial omega watch where some of the major watchmakers are trained to make omega tourbillon watches. The same team, as I understand it, is responsible for the CHRO recovery task of 18.
The 18-inch CHRO movement is wound by hand and displays the time of day as well as a 15-minute monopusher chronograph (the slider is located on the case at 6 o'clock). I accept it as true, the movement works at 2.5 Hz and has an en04ergy reserve of about forty hours. At the maximum of all methods, these movements of their refurbished form are particularly appealing and, most likely, better to look for than they have been in the past for over 100 years. In the beginning, one of these watches would probably have been used by a pilot, which makes experience due to its aviator style layout. similar dials were visible on army pilot watches, which became famous for the duration of international warfare
Each of Exquisite omega Fake Wrist Chronograph's most important wristwatches features a dial made primarily of Grand Feu enamel, inspired by the original early 20th century model. Blued steel arms on an antique sports fashion dial make for an absolutely attractive face for this piece. The case is 47.5mm wide (water resistant to 30m) in 18ct white gold with 18ct rose gold for the crown and chronograph pusher. I discovered the 20 mm brown leather band in the style of the three porthole, which is a spectacular praise for the layout. Despite the large case length, the strikingly minimized historical approaches to the watch allow the watch to be quite portable. Word the hunter-like case that is opened to expose a view of the movement inside the case, which lies on the back of a sapphire crystal.
How often did knockoff watch with steel case surpass the presentation and packaging. Vintage-grade omega branding has been converted into a used and leather-based presentation case with tools and additional straps. Omega really knows how to give away his limited editions lovingly and captivatingly. Fee for the first restrained edition of the Omega Wrist Chronograph 18 "CHRO Reference 516.52.v.30.30.00.001 is one hundred and twenty thousand CHF and the watch is part of a limited set of 18 pieces.


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Moneyweb reports:
"The media and any person has an unqualified right to access the shareholder registers of any company in South Africa; such a right is essential for effective journalism. The motive for seeking access to these registers is also totally irrelevant and the company cannot refuse access."
"These are the two main findings in the judgement of the Supreme Court of Appeal (SCA) in Moneyweb's protracted case against the Nova Property Group, Frontier Asset Management and Centro Property Group."
In the judgment, acting Judge of Appeal Fayeeza Kathree-Setiloane wrote that the three companies' interpretation of section 26(2) 'would have a negative impact on openness and transparency, and would directly undermine the work of Moneyweb, amaBhungane and other investigative journalists, as it limits the right to freedom of expression… Preventing the press from reporting fully and accurately does not only violate the rights of the journalist, but it also violates the rights of all the people who rely on the media to provide them with 'information and ideas'.
Moneyweb has been following and reporting on developments at Sharemax for a long time, and its interest continued when the three companies mentioned in the finding above took over the syndication's operations.


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luxury limo service to top golf in edison.
heading out for some golf fun? take one of our luxury limousines.
Top Golf in Edison, NJ is quickly becoming a huge favorite of our clients. It combines competitive Golf with wining and dining that is raved about. Whether your celebrating a Birthday, Anniversary, Corporate Function, Bachelor/ette Party or just heading out for the evening with friends, read on to learn more about what makes us the perfect way to get there!
the perfect way to get there.
Why choose us to go to Top Golf in Edison? Here's a few reasons we make the best choice:
- Travel together in one awesome vehicle - We have custom High Top Mercedes Limousines, Stretch Escalades and gorgeous Executive Shuttles, so no matter how big your group gets, you get there together!
Save money - Split the cost amongst your group and save on transportation. We even offer special rates for our clients heading to Top Golf! Inquire by contacting us at (732) 210-9874 today!
- Get there and back safely - Our Chauffeurs are randomly drug tested, background checked and through and through professionals. Our vehicles are world class, comfortable and clean. You may not be Tiger or Phil, but you can certainly get around like them!
bring 'em all!
don't have half of your group get stuck because they crammed into some guys ford fiesta that doesn't make it! (true story)
Go in the lap of luxury! Take our Custom Sprinter Limousine that seats up to 11 for example. Why is it so much fun? Crank up a YouTube Karaoke channel on the dual flat panel displays and blast the mega watt sound system to find out! Keep your adult beverages cold in the wet bar and enjoy during the ride! It's made for this kind of thing! Bigger group? We have everything from 21 passenger mini-bus shuttles to full blown 56 passenger Coach buses!


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Export 1 results:Author Title Type [ Year]
Filters: Keyword is User studies [Clear All Filters]
The use of mobile contact list applications and a context-oriented framework to support their design. Proceedings of the 11th International Conference on Human-Computer Interaction with Mobile Devices and Services. 79Bonn, Germany, ACM. DOI:10.1145/1613858.1613951 (125.19 KB)(2009).


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Karen as an Angel therapist facilitates healing by helping her patients get in touch with their angels .
The Angels guide her in what to say and do, channelling information for each individual to heal and guide them.
Working with energetic therapies such as Acupuncture, Body Talk and Intuitive Psychology opened Karen up to the Angel World.
As she Works she channels information and healing energies.
Karen has access to ancient information and wisdom that helps us ease our way through everyday problems.
Her studies in natural medicine give her the knowledge to work effectively on our physical and emotional problems. Working together to create harmony and balance in our health, relationships and our lives.


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There are thousand tiny things from which one can weave a beautiful little pleasures for one's life.
For me there is always that which appeals to my senses. There are certain smells that I love like fresh cut grass, flowers in spring, roses, after rain, mountain air, fresh baked bread, perfumes and scented candles and soaps. There is the joy of a feeling the smoothness and smell of washed and clean clothes.Then there is the joy of seeing the glory of nature, the sky, trees, ocean, beautiful scenic places. God's wondrous creation.
Today, I am glad I was able to do a little of my tiny enjoyment of the day, making decorative scented soaps. It's been a long time that I haven't done this. I am sorting out things here and found a box of glycerin, soap molders and essential oils, the one I use for fragrances. It's been fun!
I'm sure everyone have their own tiny enjoyments that will make your day, at least to re-route you from a busy everyday routine.


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Doodle Tuesday - Green Week
Brian Ness
For St Patrick's Day, we are posting green illustrations every day on Instagram, so you know today's doodle had to fit the theme!
here's a pair of Victorian lamps that I drew with two shades of green.
Follow LochNessMPLS for more green illustrations!
Happy Tueaday!


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There are 1 Results with the keyword : "High Assurance"
Rencontres Inria Industrie
HACL*: Verified Cryptography for Provably Secure Applications
HACL* is a new verified cryptographic library that implements popular modern cryptographic primitives. Using these primitives, HACL* implements the NaCl cryptographic API and can be used as a drop-in replacement for NaCl implementations like libsodium and TweetNaCl. The source code is C generated from F*. This implies that the verified primitives can be used as part as security critical applications and their security proofs.
Home > Centre > Paris > Innovation > RII "Les données" 17-18 octobre 2017 > Demos > HACL* : Librairie Cryptographique pour Applications à haut niveau d'assurance


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Primary school
Nursery Visits ~ for September 2016 Starters.
Nursery September 2016 Tours
Nursery Visits ~ for September 2016 Starters.
If you would like to visit our school for a nursery / early years tour please contact the office on 01438 351087. The following dates are available.
Monday 8th February 10am
Monday 8th February 2pm
Monday 22nd February 10am
Monday 22nd February 2pm
Applications open on Tuesday 9th February and close on the 18th March. You can apply online via www.hertsdirect.org/nurseryadmissions


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Beth Pickens
Beth Pickens is a Los Angeles-based consultant for artists and arts organizations. She provides career consultation, grant writing, fundraising, and financial, project, and strategic planning services for clients across the US. Before relocating to Los Angeles in 2014, Pickens was based in San Francisco and served as Senior Program Manager at Yerba Buena Center for the Arts and Managing Director of both RADAR Productions and the Queer Cultural Center.


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Back issues by year published
2005, 2004, 2003, 2002,
2001, 2000,
1999, 1998,
1997, 1996
University Communications
It was,
Boyer says, the "capstone" of his career. Although it
did not lead to further work on his part, he observes that the secretaries
at UCLA now know where to find him. He has forgiven the "leading
journal" that rejected his work. In retrospect, he says the
Nobel Prize was "the last thing I had in mind when I happened
to pick a problem with such an unusual solution," and he credits
his choice to pure luck. The other major factor that got him the
Prize, he says, was "longevity: I outlived all my competitors."
For others,
longevity may be one of the benefits of Boyer's research. He has
enhanced understanding of the kind of damage caused at the molecular
level by disease and aging. One potential application of his work
is to prevent that damage from happening to the cells of human beings.
And the
prize itself will help to bring about that, and other benefits as
well. Boyer says "one of the services of the Nobel Foundation"
is to call attention to basic science so that the importance of
new fields can be recognized. "It's a pleasure to my former
students; it gives them a boost. It makes people who've hired them
think they're better because they worked with a Nobel Prize winner."
In fact,
the research groups developed at UCLA and other American institutions
are, for Boyer, an "important return on investment that is
hardly ever recognized by the public" that pays for them. There
is "a tremendous apprentice teaching system, a relationship
between professors, graduate students and postdoctoral fellows"
that provides "the most effective tool that society could develop"
for encouraging basic science and all its beneficial consequences.


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Nitika Chopra on what it means to be "Naturally Beautiful"
Your Bella Life creator Nitika Chopra radiates health and beauty. So you'd never know that for most of her life, she struggled with a debilitating skin condition that covered her body and affected her so harshly that she was unable to walk at times.
"Even when my body was showing me that it wasn't beautiful—and really, it wasn't beautiful, it was scaly and bloody and flaky and all of that kind of stuff that happens with psoriasis—I had to figure out why I was beautiful, and that's really what fueled the whole conversation of beauty for me."
Now, she's taking that conversation on air, with her new talk show Naturally Beautiful, premiering on Veria Living on September 24 at 7:00 p.m. EST. On the show, Chopra interviews guests from all over the wellness world, like Latham Thomas, Kristin McGee, Mandy Ingber, and even the founders of Well+Good, who each address different aspects of a holistic beauty philosophy.
"I was really clear when I got the show that I did not want to do a makeup show. That's not who I am. I needed it to be an internal beauty conversation that's then reflected on the outside," says Chopra. We caught up with the glow-y New Yorker to find out more:
You talk a lot about your painful journey with psoriasis. How much did that experience influence your approach to beauty? Psoriasis influenced everything about my life, honestly. I didn't have one piece of clear skin for many years. So most of my life it was either dealing with that or dealing with a lot of conflicts, because I was always very different from everyone. Whether it was that I wasn't traditional enough, or I wasn't smart enough, or I was the only Indian among all these white people in Ohio…. The outside world was always showing me that there was something wrong with me, and then my body on top of that was showing me that there was something wrong with me. So, I had two choices. I could either believe that there was something wrong with me, or I could believe that there had to be a better way. And that's kind of what fueled everything…and the whole conversation around beauty for me.
And what does that conversation sound like now? What can we expect to hear addressed on the show? On Naturally Beautiful we talk about beauty with a holistic approach. It's very focused on beauty, but everything is through the internal lens, that's really what I'm passionate about. It's how you move and how you eat and how you think and how you take care of yourself that really affects how you are beautiful and how you feel beautiful.
So, who do you hope will watch your show? Is there a person you really want to reach? Any woman who struggles with owning how beautiful she is. It's not about what color your hair is or if your eyelashes are a certain length or whatever. The truest form of beauty is aligning with the truth of who you are. Any woman struggling with that and watching Naturally Beautiful might start to be aware of parts of themselves that are so incredibly powerful and gorgeous and amazing. We all have that within us, and I'd love for people to feel that through watching the show. —Lisa Elaine Held
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To unlock your Samsung Galaxy S8/S7/S6/S5/S4 etc. when you forget the password/pattern/PIN, don't be painful, there are three different ways to solve this problem:
Unlock your phone with Samsung's Find My Mobile, factory reset your phone in Recovery mode and the Lock Screen Removal Toolkit. In accordance with your specific cases and preference, choose one to break the lock screen.
Method 1: Unlock Samsung Galaxy with Samsung's Find My Mobile
Method 2: Hard Reset Your Samsung Galaxy Unlock your Phone
Method 3: Remove Samsung Galaxy's Lock Screen without Factory Reset
If you have previously created a Samsung account and associated the Samsung account on your phone, the "Remote control" option on your Samsung Galaxy would be turned on by default, you can unlock your Samsung Galaxy through Samsung's Find My Mobile.
Note: Check if you have enabled the "Remote control" option on your Samsung Galaxy before your phone was locked. This option is in Settings > Lock screen and security > Find My Mobile > Remote control.
Follow the steps below.
1. Visit Samsung Find My Mobile from the browser and click SIGN IN.
2. Login your Samsung account.
3. From the menu on the right side of the window, click "More" then choose "UNLOCK MY DEVICE"
4. Enter the password again then click "UNLOCK" and your Galaxy will be unlocked for you to access as you can see the icon of the lock turn green.
If you haven't logged in your Samsung account or enabled the remote control option on your Galaxy, the method 1 is useless. Factory reset is also a free way to unlock Galaxy. However, this free method will wipe out all the existing photos, messages, notes, contacts, videos etc. on your phone. You are not comparatively suggested to factory reset unless you have backed up your data previously.
1. Power off your Galaxy
2. Press and hold the Power button + Volume Down button + Home/Bixby button at the same time for a few seconds. Your phone will show up software update screen.
Just 3o seconds later it will show up another screen with big "!".
3. After about 1 minute your Samsung Galaxy will be in recovery mode.
4. Use volume buttons to move to the option "Wipe Data / Factory Reset". Press the power button to select it.
5. Once the process is complete, select "Reboot System".
As you will be upset by the data loss caused by factory reset, you need an alternative that strongly protects your files and quickly unlock your Samsung Galaxy.
Now, the Android Lock Screen Removal toolkit can efficiently remove the pattern/password lock screen without data losing. The most important thing is that you won't lose any data of your phone when unlocking the screen. Before the next steps you should first go to the website to download the toolkit.
Download WinDownload Mac
1. Install and run the toolkit. Select the feature "Lock Screen Removal" from the main window.
2. Click on the button "Start" and connect your Galaxy to computer via USB cable.
Now you should select your device model. By clicking the drop-down box you can see the models options. Once you have reconfirmed the information, click button Next.
3. The next step is entering download mode on your phone for downloading the recovery package for your phone.
Power off your Galaxy
Press and hold Volume Down + Home button + Power button at the same time until the warning screen appears after several seconds.
Press the Volume Up button to enter the Download Mode.
4. Wait for the software detects your device, next click on Remove button to start removing the lock screen.
Few This process will take a few minutes. Once the software removed the lock screen successfully your phone will restart in normal mode without any lock screen.
Download WinDownload Mac
If you can't go through the Samsung's Find My Mobile, and don't want to lose any data, choose the Lock Screen Removal tool. Even though it costs some fees, the outcome can be much satisfying.


text:
• When does technology cross the line from enabling to disabling?
As I sit here and gloat over the fact that I just got a totally legit, brand new copy of Adobe Creative Suite Master Collection for under $200, I have to ask myself: will it open doors to possibility or stand as a wall between me and my objective, which is to write, record and market my songs?
As with all technology, probably a little of both, because all tech is a blessing and a curse.
On the positive, tech enables me to do all kinds of things on my own, from designing this blog, to creating album artwork, to recording demos, to shooting photos and videos.
On the negative side, tech is seductive in that it always offers more, more, more, taking me away from my core objectives.
So as I delve into my new Adobe software, it will be incumbent on me to know when to say ENOUGH and turn a task over to someone else, budget permitting.
In fact, I think this is one of the most important things home recordists have to do: limit themselves. Because with more tracks, more plug ins, more ways to manipulate sound and images, it's way too easy to stop writing songs; it's also way too easy to delude yourself into thinking you're saving money in the long run as you do more and more on your own. Sure, your DIY chops might help you shell out fewer greenbacks overall, but in the end how much real VALUE did you get for your money? How good are your home recordings really, your home-made artwork, your Flip shot video?
No, in my opinion, do as much as you realisticallly can on your own, but through trial, error and unflinching honesty, learn when it's best to call in an expert. Otherwise, you'll spend all your time futzing around with new gizmos instead of creating.
Okay, I'm off to check out my new Adobe software (doh!).


text:
It's a action score from 1993. Academy Award Winning composer Hans Zimmer scores: "Point of No Return". The cast were Bridget Fonda, Gabriel Byrne, Dermot Mulroney, Harvey Keitel and Anne Bancroft. Hans Zimmer is a genius.
Self-Pleasure Ejaculation Rating Meter (SPERM) for Point of No Return: 92.1
That means for each 100 listenings to this soundtrack, I ejaculate from penis 92.1 times because of the music.
See my profile for more information, about SPERM formula.