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Many of the known fish herpesviruses have important aquaculture species as their natural host, and may cause serious disease and mortality. Anguillid herpesvirus 1 (AngHV-1) causes a hemorrhagic disease in European eel, Anguilla anguilla. Despite their importance, fundamental molecular knowledge on fish herpesviruses is still limited. In this study we describe the identification and localization of the structural proteins of AngHV-1. Purified virions were fractionated into a capsid-tegument and an envelope fraction, and premature capsids were isolated from infected cells. Proteins were extracted by different methods and identified by mass spectrometry. A total of 40 structural proteins were identified, of which 7 could be assigned to the capsid, 11 to the envelope, and 22 to the tegument. The identification and localization of these proteins allowed functional predictions. Our findings include the identification of the putative capsid triplex protein 1, the predominant tegument protein, and the major antigenic envelope proteins. Eighteen of the 40 AngHV-1 structural proteins had sequence homologues in related Cyprinid herpesvirus 3 (CyHV-3). Conservation of fish herpesvirus structural genes seemed to be high for the capsid proteins, limited for the tegument proteins, and low for the envelope proteins. The identification and localization of the structural proteins of AngHV-1 in this study adds to the fundamental knowledge of members of the Alloherpesviridae family, especially of the Cyprinivirus genus.
The Alloherpesviridae family, belonging to the Herpesvirales order comprises all bony fish and amphibian herpesviruses [1]. Currently, the family contains 4 genera with 11 species [2]. At least another 17 herpesviruses infecting bony fish have been described, but have not yet been sufficiently characterized to allow classification [1,3,4]. Many of these viruses cause serious disease and mortality in their respective host species, many of which are important aquaculture species. For example, channel catfish virus or Ictalurid herpesvirus 1 (IcHV-1) may cause up to 100% mortality in channel catfish (Ictalurus punctatus) fingerlings, which posed a significant problem in the big catfish aquaculture industry in the United States [5]. Koi herpesvirus or Cyprinid herpesvirus 3 (CyHV-3) is another highly contagious and virulent disease in its host species common carp and koi (Cyprinus carpio spp.), the first being one of the most economically valuable aquaculture species worldwide [6,7].
The eel herpesvirus anguillid herpesvirus 1 (AngHV-1) causes a hemorrhagic disease in the European eel, Anguilla anguilla, with increased mortality rates [8]. Because of its omnipresence in wild Western European eel stocks, AngHV-1 is regarded as one of the possible factors responsible for the decline of the wild European eel stocks since the 1980s [9]. Although the fundamental characteristics of herpesviruses of especially humans and mammals have been studied intensively, there is still little knowledge on the herpesviruses of lower vertebrates and invertebrates.
Despite their diversity in genes, host range and genome size, the virion structure is conserved throughout the entire Herpesvirales order [1]. Herpesvirus virions invariably consist of a large (diameter >100 nm) icosahedral nucleocapsid (T = 16) containing the genome, surrounded by a host-derived envelope with a diameter of about 200 nm, and an intervening proteinaceous layer called the tegument [10]. For a better understanding of the origins and replication cycle of members of the family Alloherpesviridae, the identification and characterization of the structural proteins of these alloherpesviruses is essential.
Mass spectrometry (MS) is a useful technique to identify proteins, particularly when sequence information about the protein composition is available [11]. The complete genome sequences of 5 alloherpesviruses have been determined to date and are publicly available: IcHV-1 [12], Ranid herpesvirus 1 (RaHV-1) and Ranid herpesvirus 2 (RaHV-2) [13], CyHV-3 [14] and AngHV-1 [15]. In 1995, Davison and Davison identified a total of 16 principal structural proteins for IcHV-1 by MS [16]. To enable assigning the identified proteins to the different compartments of the herpesvirus virion (i.e. capsid, tegument and envelope), complete virions were fractionated into a capsid-tegument and an envelope fraction, and premature capsids were isolated directly from infected cell nuclei. Using this approach, 4 capsid proteins, 4 envelope proteins, 5 tegument proteins and 5 tegument-associated proteins were detected for IcHV-1.
Recently, a total of 40 structural proteins were identified by MS in mature CyHV-3 particles [17]. This number resembles the total number of structural proteins reported for members of the Herpersviridae family [18-25]. It is likely that the number of structural proteins detected earlier for IcHV-1 is an underrepresentation of the actual number, caused by the limited sensitivity of MS at the time. The CyHV-3 structural proteins were assigned to the different herpesvirus compartments on the basis of sequence homology and bioinformatics [17]. Since sequence homology between CyHV-3 and IcHV-1 is limited, the putative location in the virion of the majority of the identified proteins could not be assigned.
The current study aimed at identifying and characterizing the structural proteins of AngHV-1. The approach in fact entails a combination of the capsid retrieval and virion fractionation techniques previously used for IcHV-1 [16], and the high sensitivity liquid chromatography tandem mass spectrometry (LC-MS/MS) approach used for CyHV-3 [17]. The envelope proteins were further characterized using bioinformatics. The results of this study not only provide insight into the protein composition of the mature extracellular AngHV-1 virions, but also give a first indication of the conservation of structural proteins within the Alloherpesviridae family.
Materials and methods
Production and purification of AngHV-1 virions
The Dutch AngHV-1 strain CVI500138 [26] was isolated and propagated in monolayers of eel kidney (EK-1) cells [27] in 150 cm2 cell culture flasks infected at a multiplicity of infection of 0.1 as described previously [15]. Virions were purified from the culture medium using a previously described protocol with some modifications [28]. Three days post-infection, cell culture medium containing cell-released mature virions was collected and cleared from cell debris by centrifugation at 3 500 × g for 20 min at 4°C (Hermle Labortechnik Z400K, Wehingen, Germany). From here on virus was kept on ice. Virus was pelleted by ultracentrifugation at 22 000 rpm for 90 min at 4°C, with slow acceleration and slow deceleration (Beckman Coulter Optima L70K Ultracentrifuge with rotor SW28, Brea, CA, USA). The pellet was resuspended in 1 ml TNE buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH = 7.5) by pipetting and vortexing. The virus suspension was layered onto a 10 to 60% linear sucrose gradient in TNE buffer. Following ultracentrifugation (rotor SW41Ti, 22 000 rpm for 18 h at 4°C), the virus band was collected. Subsequently, the virus was washed in 10 volumes of TNE buffer and concentrated by ultracentrifugation (rotor SW41Ti, 30 000 rpm for 3 h at 4°C). The virus pellet was resuspended in 200 μL TNE buffer and stored at -80°C until further use.
Fractionation of AngHV-1
Lipid envelopes were released from the capsid-teguments by incubation with a nonionic detergent as described previously [16]. Briefly, an equal volume of solubilization buffer (50 mM Tris-HCl, 0.5 M NaCl, 20 mM EDTA, 2% (v/v) Nonidet P40) was added to the virus solution, incubated on ice for 15 min, and microcentrifuged at 25 000 × g for 5 min at 4°C (Eppendorf 5417R, Hamburg, Germany). Supernatant containing the envelopes was collected by pipetting. The capsid-tegument pellet was washed by vortexing in 100 μL of ice-cold 0.5x solubilization buffer followed by microcentrifugation at 25 000 × g for 5 min at 4°C. The supernatant was discarded by pipetting, 50 μL of cold TNE-buffer was added, and the pellet was resuspended by probe sonication (MSE, London, UK) for 10 s. The envelope fraction was clarified further by three subsequent microcentrifugation steps (25 000 × g for 5 min at 4°C), each time collecting the supernatant by decantation. The capsid-tegument and envelope fractions were stored at -80°C until further use.
Purification of AngHV-1 capsids
AngHV-1 infected EK-1 cells were washed with PBS to remove complete virus particles. Cells from 4 150 cm2 cell culture flasks were collected by scraping using a rubber policeman in 9 mL ice-cold TNE buffer. Cells were lysed by adding 1 mL of 10% (v/v) Triton X-100 in TNE (final concentration 1% (v/v) Triton X-100) and probe sonication on ice for three times 20 s. Debris was pelleted by ultracentrifugation (rotor SW41Ti, 10 000 rpm for 10 min at 4°C). Capsids were purified by sucrose cushion (40% in TNE) ultracentrifugation (20 000 rpm for 1 h at 4°C). The pellet was resuspended in 0.5 mL TNE and further purified by centrifugation on a linear 10-60% sucrose gradient in TNE for 1 h at 20 000 rpm. The two resulting bands, presumably containing capsids, were separately collected as an upper and a lower band, washed in TNE buffer and pelleted by ultracentrifugation (20 000 rpm for 1 h at 4°C). The supernatant was discarded, the capsid pellets resuspended in 200 μL TNE buffer and stored at -80°C until further use.
Electron microscopy
Nickel grids (400-mesh) with a carbon-coated collodion film were placed upside down on a drop of complete virion suspension, virion fraction suspension or capsid suspension, and incubated for 10 min. After incubation, grids were washed with distilled water and stained with 2% phosphotungstic acid (pH = 6.8). Grids were examined with a Philips CM10 transmission electron microscope (Amsterdam, The Netherlands).
Proteins in purified virions, virion fractions and capsids were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Virions in TNE buffer were mixed 1 : 1 with denaturizing sample buffer containing dithiothreitol (DTT) and heated for 5 min at 95°C. Samples were loaded onto 12% NuPAGE Novex Bis-Tris gels (Invitrogen by Life Technologies, Carlsbad, CA, USA) and ran for 2 h at 80 V in NuPAGE MOPS SDS-running buffer (Invitrogen). Gels were stained with Coomassie blue R-250 (Merck, Whitehouse Station, NJ, USA) or Silver (PlusOne Silver Staining Kit, GE Healthcare, Chalfont St. Giles, UK).
LC-MS/MS approach
The capsid proteins in the upper band were analyzed by SDS-PAGE and stained with Coomassie blue. The five visible protein bands were collected separately in gel slices. The gel segments were incubated in 10 mM DTT in 50 mM ammonium bicarbonate (ABC) buffer at 60°C for 1 h to reduce disulfide bridges and subsequently in 100 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO, USA) in ABC buffer at room temperature for 1 h in the dark. After a final wash step with ABC buffer, the gel material was dried. Trypsin digestion was performed as described previously by Ince et al. [29]. In short, in-gel protein digestion was performed using sequencing grade modified porcine trypsin (Promega, Madison, WI, USA) in ABC buffer (10 ng/μL). After incubation overnight, samples were bath sonicated, and after centrifugation the basic supernatants were collected. The remaining gel pieces were extracted with 10% triflouroacetic acid (TFA), followed by 5% TFA, followed by 15% acetonitrile/1% TFA. The latter extracts were combined with the supernatants of the original digests, vacuum-dried, and dissolved in 20 μL 0.1% formic acid in water. The peptides resulting from this digestion were analyzed by LC-MS/MS as described previously [29].
1D gel/nanoLC-MS/MS approach
Proteins from purified virions and from the three virion fractions (capsid, envelope and capsid-tegument) were separated by SDS-PAGE on 4-20% acrylamide 7 cm gels (Invitrogen) and stained with Coomassie blue. Separated proteins in the gel were excised in 20 and 30 serial slices along the lane, for complete virions and virion fractions, respectively. Gel slices were submitted to in-gel digestion with sequencing grade modified trypsin as described previously [17]. Briefly, gels were washed successively with ABC buffer and ABC buffer/acetonitrile (ACN) 50% (v/v). Proteins were reduced and alkylated using DTT and iodoacetamide followed by washing with ABC and ABC/ACN. Resulting peptides were analyzed by 1D gel/nanoLC-MS/MS using a 40 min ACN gradient as described by Mastroleo et al. [30].
2D nanoLC-MS/MS approach
Only proteins of purified complete virions were submitted to 2D nanoLC-MS/MS analysis. Proteins were extracted from complete virions using guanidine chloride (GC) as described previously [17]. In short, the virions were suspended in 6 M GC and sonicated for 5 min and shaken at 900 rpm for 30 min at room temperature. After centrifugation the proteins were reduced with 10 mM DTT at 60°C for 30 min and alkylated with 25 mM iodoacetamide at 25°C for 30 min in the dark. Proteins were recovered by acetone precipitation and dissolved in 50 mM Tris/HCl (pH = 8), 2 M urea. The proteins were digested overnight at 37°C with trypsin (enzyme : substrate ratio = 1 : 50). Tryptic peptides were cleaned using spin tips (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Proteins were analyzed by 2D (strong cation exchange, reverse-phase) chromatography and online MS/MS, as described by Mastroleo et al. [30] except that only 3 salt plugs of 25, 100 and 800 mM NH4Cl were analyzed in addition to the SCX flow through.
MS/MS analyses
Peptides were analyzed using an HCT ultra ion Trap (Bruker, Billerica, MA, USA). Peptide fragment mass spectra were acquired in data-dependent AutoMS(2) mode for 4 most abundant precursor ions in all MS scan. After acquisition of 2 spectra, precursors were actively excluded within a 2 min window, and all singly charged ions were excluded. Data were processed using Mascot Distiller with default parameters. An in-house Mascot 2.2 server (Matrix Science, London, UK) was used for dataset searching against the NCBI Alloherpesviridae database. The default search parameters used were the following: Enzyme = Trypsin; Maximum missed cleavages = 2; Fixed modifications = Carbamidomethyl (C); Variable modifications = Oxidation (M); Peptide tolerance ± 1.5 Dalton (Da); MS/MS tolerance ± 0.5 Da; Peptide charge = 2+ and 3+; Instrument = ESI-TRAP. Only sequences identified with a Mascot Score greater than 30 were considered, which indicates identity or extensive homology (p-value < 0.05). Single peptide identification was systematically evaluated manually. The exponentially modified protein abundance index (emPAI) [31] was calculated to estimate protein relative abundance for the complete virion extracts. The protein abundance index (PAI) is defined as the number of observed peptides divided by the number of observable peptides per protein. The exponentially modified PAI (10PAI - 1) is proportional to protein content in a protein mixture in LC-MS/MS experiments.
The amino acid sequences of all identified AngHV-1 structural proteins were analyzed using bioinformatic tools from the CBS website [32] to identify potential transmembrane domains (TMHMM) [33], signal peptides (SignalP) [34], and glycosylation sites (NetNGlyc [35] and NetOGlyc [36]).
Results and Discussion
Electron microscopy
Purified virus, fractionated virus, and purified capsids were checked for quality by transmission electron microscopy (EM, pictures not shown). The preparation of purified virus contained complete virions with intact or disrupted envelope, capsids and envelopes. The upper band of sucrose gradient purified capsids primarily contained capsids with an electron-lucent inner appearance, while the lower band showed capsids with an electron-dense core. No cell debris was seen in the capsid fractions. In the capsid-tegument fraction, only capsids and no envelopes were visible. The envelopes in the envelope fraction were largely unrecognizable and present as clusters of membrane remnants. Only very few capsids contaminated the envelope fraction. Overall, the virus and capsid purification as well as the virus fractionation could be considered successful, at least as evaluated by EM.
Davison and Davison [16] already mentioned for IcHV-1 that the resulting upper and lower bands after capsid purification parallel the density separation of mammalian herpesvirus capsids into A, B and C forms (in order of increasing density) as initially described by Gibson and Roizman [37]. The lower band consists of capsids comparable to the mature DNA-containing C form capsids, while the upper band comprises both the immature DNA-lacking B form capsids (containing additional core proteins) and the erroneous DNA-lacking A form capsids. For the sake of clarity in this paper we will follow the nomenclature as initially proposed for the IcHV-1 capsids: U capsids for the capsids found in the upper (U) band and L capsids for the capsids found in the lower (L) band [16]. The L capsid fraction contained significantly more DNA-containing capsids than the U capsid fraction (data not shown). This was in agreement with earlier observations on DNA content of premature mammalian herpesvirus capsids [38,39], and with the current view of herpesvirus capsids being first assembled around a scaffold with the DNA being inserted later [40].
Proteins in purified virions, capsids and virion fractions were analyzed by SDS-PAGE to check purity and analyze the protein content of the different virion compartments. Thirty-five bands were visible for the complete virions using silver staining (Figure 1). Purified L capsids showed 4 clear bands, the U capsids showed an additional fifth protein band of low molecular weight not present in complete virus particles (Figure 1a). The envelope fraction resulted in 20 proteins (Figure 1b). The capsid-tegument fraction resulted in a smear in the high molecular weight region and at least 27 individual proteins could be differentiated. All four L capsid proteins were also present in the capsid-tegument fraction. Several proteins were predominant in either the envelope-tegument or the capsid-tegument fraction, but at least 8 proteins were clearly present in both fractions, presumably representing tegument-associated proteins.
Figure 1. One dimensional SDS-PAGE profile of different AngHV-1 virion fractions. Loaded onto 12% Bis-Tris polyacrylamide and silver stained: 1a) proteins in the purified complete virions (lane 1), L capsids (lane 2) and U capsids (lane 3), numbered arrows indicate the excised protein bands which were analyzed by LC-MS/MS (Table 1); 1b) proteins in the purified complete virions (lane 4), envelope fraction (lane 5), and capsid-tegument fraction (lane 6). Molecular masses (kDa) are indicated on the left.
AngHV-1 capsid proteins
The five U capsid proteins were excised from a Coomassie blue stained gel and identified by LC-MS/MS. The proteins were identified as the major capsid protein (ORF104), the proteins encoded by ORF48 and ORF42, and the capsid triplex protein 2 (ORF36) (Table 1). The fifth protein, which did not seem to be present in the L capsids, appeared to be the capsid protease-and-scaffolding protein (ORF57). Indeed, in mammalian herpesviruses, this protein serves as a scaffold around which the capsid is built, and is proteolytically cleaved and extruded at the moment of DNA incorporation in the capsid [40].
Table 1. Capsid proteins of AngHV-1 as identified by 1D gel/nanoLC-MS/MS in purified U capsids
Based on size and relative abundance compared to the capsid triplex protein 2 and the major capsid protein, AngHV-1 ORF42 is the best candidate to encode the capsid triplex protein 1 (expected ratio 1 : 2 : 3 [37,41]). ORF42 shows no convincing sequence homology with its putative functional homologue in IcHV-1 ORF53 [16]. It shows sequence homology, however, with ORF66 of the more closely related CyHV-3, encoding an abundant structural protein [17]. This low sequence conservation of the capsid triplex protein 1 is comparable with the sequence conservation among the capsid proteins of members of the Herpesviridae family [16]. Another highly abundant and large capsid protein is encoded by AngHV-1 ORF48, for which sequence homology was absent, however.
To obtain complementary data another gel loaded with U capsid proteins separated by SDS-PAGE was divided into 30 serial slices which were analyzed with 1D gel/nanoLC-MS/MS. This analysis resulted in the identification of another 6 low abundant proteins, of which 4 were contaminating other high abundant structural proteins, and 2 were additional putative capsid proteins. For the protein encoded by the spliced ORF100, 9 peptides were found in the U capsid fraction. Its high conservation among other alloherpesviruses hints to a possibly important but yet unknown function. The protein encoded by AngHV-1 ORF126 was little abundant (only 2 peptides in the U capsid fraction) and is not conserved in other herpesviruses. The overall high rate of capsid protein conservation (5 out of 7) was comparable with that of members of the Herpesviridae family and resembles the functional conservation of capsid structure in the Herpesvirales order.
AngHV-1 envelope proteins
Removal of virus envelopes by treatment with a non-ionic detergent has allowed proteins to be assigned as components of the envelope versus capsid-tegument [16]. Proteins were principally defined as envelope proteins when present in the envelope fraction in higher concentrations (based on their emPAI) than in the capsid-tegument fraction, and when having certain characteristics of membrane proteins. A total of 30 proteins were identified by 1D gel/nanoLC-MS/MS in this sample and based on the former criteria 10 were annotated as putative envelope proteins (Table 2). Sixteen of the 20 non-envelope proteins were putative tegument proteins (Table 3), 4 were putative capsid proteins and little abundant. Eight of the envelope proteins comprised both a transmembrane domain and a signal peptide or anchor. The proteins encoded by ORF8 and ORF108 lacked a signal peptide, but were exclusively detected in the envelope fraction and not in the capsid-tegument fraction. The presumed multiple transmembrane protein encoded by AngHV-1 ORF49 was only detected in very low abundance in complete virion preparations and not in one of the virion fractions. The CyHV-3 homologue of this protein (ORF83) was not detected in CyHV-3 virions [17], possibly due to its low abundance. A signal peptide was predicted for only one other structural protein of AngHV-1 (encoded by ORF103), but not a transmembrane domain. Moreover, this protein was exclusively found in the capsid-tegument fraction, indicating that it is a putative tegument protein and not an envelope protein.
Table 2. Envelope proteins of AngHV-1 as identified by 1D gel/nanoLC-MS/MS in the envelope fraction
Table 3. Tegument proteins of AngHV-1 as identified by 1D gel/nanoLC-MS/MS in the capsid-tegument and envelope fraction
The two ORF encoding the most abundant envelope proteins demonstrated interesting sequence homologies. The most abundant AngHV-1 envelope protein is encoded by ORF51, which shows low sequence homology with CyHV-3 ORF81 (E-value = 10-4) in a directed search against members of the Alloherpesviridae family. CyHV-3 ORF81 encodes an abundant multiple transmembrane protein, thought to be the immunodominant envelope protein of CyHV-3 [42]. CyHV-3 ORF81 is the positional homologue of IcHV-1 ORF59, the latter being the major envelope protein [16]. While the homologue of this protein also seems to be a major envelope protein in AngHV-1, only a few peptides of this protein were found in CyHV-3 virions [17].
The second most abundant AngHV-1 envelope protein, encoded by ORF95, was previously shown to be related to the haemagglutinin-esterase protein of infectious salmon anaemia virus (ISAV) (E-value = 10-16) [15]. In the piscine orthomyxovirus ISAV this viral surface glycoprotein is responsible for viral attachment and release [43]. Herpesviruses are known to be capable of gene capture from cells or other viruses. Likewise AngHV-1 ORF95 might originate from ISAV or, for example, from a yet uncharacterized eel orthomyxovirus (Olga Haenen, personal communication).
AngHV-1 ORF67, encoding a large envelope protein, was previously shown to exhibit sequence homology with the White bream virus (WBV) spike protein (E-value = 10-24) [15], a protein probably mediating receptor binding and fusion between viral and cellular membranes [44]. A search for the yet unidentified AngHV-1 homologue of the presumed major glycoprotein present in other alloherpesviruses (IcHV-1 ORF46 [12], CyHV-3 ORF99 [14], RaHV-1 ORF46 [13] and RaHV-2 ORF72 [13]) resulted in low sequence homology of AngHV-1 ORF67 with RaHV-1 ORF46 (E-value = 10-3) in particular. The latter finding raises questions about the origin of AngHV-1 ORF67. One option might be that the current AngHV-1 ORF67 is the result of gene capture from WBV and subsequent genetic reassortment with the original major glycoprotein.
Human herpesviruses carry between 12 and 20 viral membrane proteins in their envelope [45]. For the alloherpesviruses CyHV-3 and AngHV-1, a total of 13 [17] and 11 (this paper) viral envelope proteins were predicted, respectively. Despite their large genomes, these alloherpesviruses seem to encode a relatively low number of membrane proteins. Many envelope proteins are specific to each herpesvirus type. However, among the members of the Herpesviridae family five glycoproteins are broadly conserved, namely gB, gH, gL, gM and gN [45]. Glycoprotein gB and a complex formed by gH and gL are involved in the fusion of the viral envelope and plasma membrane. Glycoproteins gM and gN form another complex. The fact that only two envelope proteins seem to be conserved among the members of the Alloherpesviridae family, is yet another indication that the evolutionary distance among fish and amphibian herpesviruses is greater than among mammalian, bird and reptile herpesviruses.
AngHV-1 tegument proteins
Comparison of the proteins found in the capsid-tegument fraction with the proteins identified in purified capsids allowed the identification of the tegument proteins. Tegument proteins were defined as proteins found in the capsid-tegument fraction, but not (or only in trace amounts) in the purified capsid fraction, and not meeting the criteria as defined for envelope proteins. A total of 32 proteins were identified by 1D gel/nanoLC-MS/MS in the capsid-tegument sample, 22 of which were considered to be tegument proteins (Table 3). Six tegument proteins were exclusively found in the capsid-tegument fraction and for two other capsid-tegument proteins (ORF19 & ORF40) relatively high amounts were detected in the capsid-tegument fraction and relatively low amounts in the envelope fraction. Hence these proteins were called tegument proteins. The majority (14) of the proteins in the capsid-tegument fraction was, however, also found in the envelope fraction, indicating that these proteins are rather loosely bound to the tegument. These proteins were therefore classified as tegument-associated proteins. This distinction between true tegument and tegument-associated proteins is arbitrary and presumably depends on the fractionation conditions used. A more precise differentiation would be based on the interaction of the tegument proteins with either the capsid ("inner tegument") or the cytoplasmic domains of viral envelope proteins ("outer tegument") [40,46].
Nine sequence similarity based gene families have been identified within the AngHV-1 genome [15]. Most of these ORF encode proteins with unknown functions. Three of the four proteins of the ORF13 gene family were identified as tegument-associated proteins, indicating that this gene family encodes proteins with a tegument-related function.
Among the 22 tegument(-associated) proteins found, 10 showed sequence homology with CyHV-3 ORF, which therefore can now be classified as presumed tegument proteins. The largest AngHV-1 tegument-associated protein, ORF83, contains a cysteine protease domain in the N-terminal region homologous to the Ovarian Tumor gene in Drosophila [47]. This domain is also found in CyHV-3 ORF62 and IcHV-1 ORF65. Based on its size and conservation, this ORF has been suggested by Michel et al. [17] to encode the homologue of the large tegument protein UL36, which is conserved among the members of the Herpesviridae family [46,48]. UL36 is an essential and abundant structural polypeptide with multiple functions. It binds to the capsid on the one hand, and to several major tegument components on the other hand, suggesting that UL36 plays an important role in the structural organization of the tegument. A putative function could not be determined for any of the other AngHV-1 tegument(-associated) proteins.
Except for the large tegument protein, no significant sequence homology of tegument proteins was found with any of the other alloherpesviruses. This suggests that conservation of the tegument proteins is high between closely related alloherpesviruses, but hardly present throughout the whole family. This level of conservation reflects the greater divergence of the Alloherpesviridae family compared to the Herpesviridae family, among which almost a dozen tegument proteins are conserved [46,49,50]. More sequence data from other alloherpesviruses and functional characterization of yet identified tegument proteins is necessary to determine the exact level of tegument protein conservation [45].
AngHV-1 virion composition
In order to unravel the relative abundance of the structural proteins of AngHV-1, proteins from purified virions separated by SDS-PAGE were excised from the gel in 20 serial slices. After in-gel digestion, the resulting peptides were analyzed by 1D gel/nanoLC-MS/MS. An in-solution trypsin digest MS approach, termed 2D nanoLC-MS/MS, was used as a complement to this gel based LC-MS/MS approach to enhance the recovery of peptides derived from proteins that are prone to aggregation, contain hydrophobic peptides, or are present at low abundance. The 1D gel and 2D nanoLC-MS/MS analyses of complete virions resulted in the identification of 33 and 27 viral proteins respectively, with an overlap of 26 proteins (Table 4). All but one of these proteins had already been identified in the virion fractions. AngHV-1 ORF49, presumably encoding a type 3 membrane protein, was detected in very low abundance in both complete virion analyses.
Table 4. Structural proteins of AngHV-1 as identified by 1D gel and 2D nanoLC-MS/MS
For the 1D gel and 2D nanoLC-MS/MS analyses of complete virions the emPAI was calculated to estimate protein relative abundance (Table 4). The emPAI from both analyses is generally comparable. A schematic representation of the AngHV-1 virion was made based on predicted protein mass, localization of the structural proteins of AngHV-1 as determined by analyses of the respective fractions, and the relative abundance in complete virions as determined for the protein extract analyzed by 1D gel/nanoLC-MS/MS (Figure 2).
Figure 2. Schematic representation of the protein composition of mature extracellular AngHV-1 virions. The typical herpesvirus compartments are indicated as a hexagon (capsid), circle (envelope) and the space in between (tegument). The location of the structural proteins is indicated. Proteins identified as true tegument proteins directly surround the capsid, proteins identified as tegument-associated proteins are located in the outer part of the tegument. The orientation of the envelope proteins indicates the number of transmembrane domains. The predicted protein mass is logarithmically indicated in size. The relative abundance (emPAI) as determined for the 1D gel/nanoLC-MS/MS analyses of complete virions is indicated in color intensity (see scale).
Host proteins associated with AngHV-1 virions
Several cellular host proteins end up in mature herpesvirus virions, either intentionally or accidentally in the process of virus assembly and release from the cell. We also performed searches for non-viral host-originating proteins for all LC-MS/MS datasets acquired in this study. The proteins were identified by searching the peptides against a bony vertebrate database, since there are only very few genomic Anguilla spp. sequences available. In the 1D gel and 2D nanoLC-MS/MS analyses of complete virions, 30 and 15 host proteins were detected, respectively, with an overlap of 3 proteins (Additional file 1). A total of 28 unique host proteins associated with AngHV-1 virions were found, compensating for the several hits against protein homologues in different (fish) species. This number is somewhat higher than the number of host proteins found to be associated with CyHV-3 [17]. It resembles numbers found for several mammalian herpesviruses, either cushion [23,25] or gradient purified [24], however.
Additional file 1. Table S1: Host proteins associated with AngHV-1 virions as identified by 1D gel/nanoLC-MS/MS and 2D nanoLC-MS/MS. This table contains the non-viral host-originating proteins identified in the 1D gel and 2D nanoLC-MS/MS analyses of complete virions by searching the peptides against a bony vertebrate database. Thirty and 15 host proteins were detected, respectively, with an overlap of 3 proteins, representing a total of 28 unique host proteins associated with AngHV-1 virions.
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The host proteins found to be associated with AngHV-1 virions include cytoskeleton proteins (α-actin, β-actin, actin-depolymerization factor, filamin, keratin, profilin, septin, etc.), proteins involved in transport (fatty acid binding proteins, lipocalin, myelin), an adrenoreceptor-like protein, proteins involved in glycolysis (aldolase and glyceraldehyde-3-phosphate dehydrogenase) and protein glycosylation, regulatory proteins (ubiquitin and a WD repeat containing protein), a protein involved in translation control (Sp5 transcription factor), proteins involved in the immunological response (pentraxin) and stress-response (heat shock proteins 70 & 90), and several proteins with yet unknown functions.
Several of the classifiable virion associated host proteins have also been described in CyHV-3 and mammalian herpesviruses [16-20,22-25]. The composition of the set of incorporated host proteins might, however, be influenced by the type of cell culture used for virus propagation. All virion associated host proteins were found in low concentrations of only 1 to a maximum of 18 peptides per protein (Additional file 1), suggesting that these proteins are only minor components of the virion. Although many of the identified host proteins have previously been associated with herpesvirus virions, it is possible that some of these proteins represent minor cellular contaminants of the virion preparations.
Evaluation of the approach
In this study, three approaches were followed for the detection of the structural proteins of AngHV-1. The first and most straight-forward method was the in-solution trypsin digest MS approach termed 2D nanoLC-MS/MS. A total of 27 unique structural proteins were identified. The second approach was the extraction and separation of AngHV-1 virion proteins by SDS-PAGE, followed by excision of 20 contiguous sections of the gel along the migration path, in-gel trypsin digestion and subsequent nanoLC-MS/MS analysis. This approach resulted in the identification of another 7 unique AngHV-1 structural proteins. The third approach, which intended to assign the identified proteins to the different AngHV-1 virion compartments, resulted in the identification of another 6 structural proteins. The proteins detected by only one of the approaches were generally little abundant.
A total of 40 structural proteins of AngHV-1 were identified (Tables 1, 2, 3 and 4). This number is not likely to represent the actual total number of AngHV-1 structural proteins, but will nevertheless represent the majority. For the related CyHV-3 virion, a similar number of structural proteins was recently identified, but Michel et al. used several additional protein extraction methods before separating the virions by SDS-PAGE [17]. Based on their results we decided to use the two most efficient extraction and separation procedures for the current study, namely GC extraction followed by in-solution trypsin digestion for the 2D nanoLC-MS/MS approach, and protein extraction with SDS followed by separation by SDS-PAGE and in-gel trypsin digestion for the 1D gel/nanoLC-MS/MS. Higher virion concentrations as well as additional analyses of separate virion fractions significantly contributed to the recovery of peptides derived from proteins present in low abundance.
When Davison and Davison identified the structural proteins of IcHV-1 in 1995, they found a total of 16 principal structural proteins [16]. We followed their capsid retrieval and virion fractionation techniques, confirmed by EM and SDS-PAGE, but used a more sensitive MS technique. The number of structural proteins found for AngHV-1 was significantly higher than the number found earlier for IcHV-1. Generally, the capsid retrieval and virion fractionation techniques worked well in combination with LC-MS/MS. Nevertheless, several highly abundant proteins showed some overspill in fractions where the proteins were not expected to be. This probably indicates that the virion fractionation techniques were not conclusive. In addition, it could be possible that proteins from which only one or two peptides were found are actually contaminating high abundant non-structural proteins, detected by the highly sensitive LC-MS/MS technique used.
Several observations support the accuracy of the final results of our approach. First, the number of proteins found in the different fractions was highly comparable to those determined by recent comprehensive characterization of extracellular herpes simplex virus type 1 and pseudorabies virus virions [23,25]. Second, all AngHV-1 sequence similarity based homologues of CyHV-3 structural proteins were found in AngHV-1 virions. Third, the proteins with a sequence predicted function were found in the expected fractions. Fourth, as for the low abundant proteins, several of these proteins have homologues in CyHV-3 and were accordingly detected in CyHV-3, which were purified using a different protocol [17]. Fifth, all proteins identified as envelope proteins showed many if not all of the basic characteristics of an envelope protein, whilst none of the other structural proteins showed a predicted transmembrane domain, and a signal peptide was predicted for only one non-envelope protein. Sixth, non-structural proteins such as proteins involved in DNA replication, DNA packaging or presumably secreted immunomodulatory proteins were not identified.
The identification and localization of the structural proteins of AngHV-1 in this study adds to the fundamental knowledge of members of the Alloherpesviridae family, especially for the known members of the related Cyprinivirus genus. The localization and putative function of the identified AngHV-1 structural proteins can now be extrapolated for homologous genes in other alloherpersviruses. In addition, the results presented give a first indication of the conservation of structural proteins within the Alloherpesviridae family. Conservation is high in the capsid fraction, limited in the tegument fraction and low in the envelope fraction. In this respect the Alloherpesviridae family resembles the Herpesviridae family, yet the evolutionary distance among fish and amphibian herpesviruses is significantly greater than among mammalian, bird and reptile herpesviruses. For AngHV-1 in particular, the results of this study will facilitate more directed functional characterization of proteins of interest. Moreover, this information is essential in further studies on the pathobiology of this virus, and will support the development of specific diagnostic tools and vaccines.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SJvB designed the study, carried out the virus culture, virion purification & fractionation, capsid isolation, SDS-PAGE, sample preparations for LC-MS/MS, bioinformatic analyses and drafted the manuscript. BL performed the 1D gel/nanoLC-MS/MS and 2D nanoLC-MS/MS measurements of complete virions, capsids and virion (including sample preparations), supervised by RW and coordinated by AV. OLMH headed the cell and virus cultures. SB assisted in the sample preparations for LC-MS/MS and performed the LC-MS/MS analyses of the U capsids, supervised by JJMV. BPHP, PJMR and AV advised on the study design and manuscript. MYE coordinated the study. All authors read and approved the final manuscript.
We are grateful to Ineke Roozenburg and Michal Voorbergen-Laarman (Central Veterinary Institute (CVI) of Wageningen UR) for technical support with cell and virus culture, to Lisette Ruuls (CVI) for EM sample preparations and guidance with the EM, and to Karel Riepema (CVI) for technical assistance with SDS-PAGE and gel staining. We thank Andrew Davison (MRC-University of Glasgow Centre for Virus Research) for useful comments on premature capsid purification and virion fractionation. This work was supported by a grant from the University of Liège (Crédit d'Impulsion) and a FRFC grant from the FNRS (2.4622.10).
Davison AJ, Eberle R, Ehlers B, Hayward GS, McGeoch DJ, Minson AC, Pellett PE, Roizman B, Studdert MJ, Thiry E: The order Herpesvirales.
Arch Virol 2009, 154:171-177. PubMed Abstract | Publisher Full Text
Virus Taxonomy: 2009 Release [http://www.ictvonline.org/virusTaxonomy.asp?version=2009] webcite
Waltzek TB, Kelley GO, Alfaro ME, Kurobe T, Davison AJ, Hedrick RP: Phylogenetic relationships in the family Alloherpesviridae.
Dis Aquat Organ 2009, 84:179-194. PubMed Abstract
Jakob NJ, Kehm R, Gelderblom HR: A novel fish herpesvirus of Osmerus eperlanus.
Virus Genes 2010, 41:81-85. PubMed Abstract | Publisher Full Text
Plumb JA: Channel Catfish Herpesvirus. In Viruses of Lower Vertebrates. Edited by Ahne W, Kurstak E. Berlin: Springer-Verlag; 1989:198-216.
Haenen OLM, Way K, Bergmann SM, Ariel E: The emergence of Koi herpesvirus and its significance to European aquaculture.
Michel B, Fournier G, Lieffrig F, Costes B, Vanderplasschen A: Cyprinid herpesvirus 3.
Emerg Infect Dis 2010, 16:1835-1843. PubMed Abstract | Publisher Full Text
Haenen OLM, Dijkstra SG, Tulden PW, Davidse A, van Nieuwstadt AP, Wagenaar F, Wellenberg GJ: Herpesvirus anguillae (HVA) isolations from disease outbreaks in cultured European eel, Anguilla anguilla in the Netherlands since 1996.
Haenen OLM, Lehmann J, Engelsma MY, Stürenberg FJ, Roozenburg I, Kerkhoff S, Klein Breteler J: The health status of European silver eels, Anguilla anguilla, in the Dutch River Rhine Watershed and Lake IJsselmeer.
Aquaculture 2010, 309:15-24. Publisher Full Text
Booy FP, Trus BL, Davison AJ, Steven AC: The capsid architecture of channel catfish virus, an evolutionarily distant herpesvirus, is largely conserved in the absence of discernible sequence homology with herpes simplex virus.
Virology 1996, 215:134-141. PubMed Abstract | Publisher Full Text
Pandey A, Mann M: Proteomics to study genes and genomes.
Nature 2000, 405:837-846. PubMed Abstract | Publisher Full Text
Davison AJ: Channel catfish virus: a new type of herpesvirus.
Virology 1992, 186:9-14. PubMed Abstract | Publisher Full Text
Davison AJ, Cunningham C, Sauerbier W, McKinnell RG: Genome sequences of two frog herpesviruses.
J Gen Virol 2006, 87:3509-3514. PubMed Abstract | Publisher Full Text
Aoki T, Hirono I, Kurokawa K, Fukuda H, Nahary R, Eldar A, Davison AJ, Waltzek TB, Bercovier H, Hedrick RP: Genome sequences of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide.
J Virol 2007, 81:5058-5065. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
van Beurden SJ, Bossers A, Voorbergen-Laarman MH, Haenen OL, Peters S, Abma-Henkens MH, Peeters BP, Rottier PJ, Engelsma MY: Complete genome sequence and taxonomic position of anguillid herpesvirus 1.
J Gen Virol 2010, 91:880-887. PubMed Abstract | Publisher Full Text
Davison AJ, Davison MD: Identification of structural proteins of channel catfish virus by mass spectrometry.
Virology 1995, 206:1035-1043. PubMed Abstract | Publisher Full Text
Michel B, Leroy B, Stalin Raj V, Lieffrig F, Mast J, Wattiez R, Vanderplasschen A, Costes B: The genome of cyprinid herpesvirus 3 encodes 40 proteins incorporated in mature virions.
J Gen Virol 2010, 91:452-462. PubMed Abstract | Publisher Full Text
Varnum SM, Streblow DN, Monroe ME, Smith P, Auberry KJ, Pasa-Tolic L, Wang D, Camp DG, Rodland K, Wiley S, Britt W, Shenk T, Smith RD, Nelson JA: Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome.
J Virol 2004, 78:10960-10966. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kattenhorn LM, Mills R, Wagner M, Lomsadze A, Makeev V, Borodovsky M, Ploegh HL, Kessler BM: Identification of proteins associated with murine cytomegalovirus virions.
J Virol 2004, 78:11187-11197. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Johannsen E, Luftig M, Chase MR, Weicksel S, Cahir-McFarland E, Illanes D, Sarracino D, Kieff E: Proteins of purified Epstein-Barr virus.
Proc Natl Acad Sci USA 2004, 101:16286-16291. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
O'Connor CM, Kedes DH: Mass spectrometric analyses of purified rhesus monkey rhadinovirus reveal 33 virion-associated proteins.
J Virol 2006, 80:1574-1583. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Dry I, Haig DM, Inglis NF, Imrie L, Stewart JP, Russell GC: Proteomic analysis of pathogenic and attenuated alcelaphine herpesvirus 1.
J Virol 2008, 82:5390-5397. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kramer T, Greco TM, Enquist LW, Cristea IM: Proteomic characterization of pseudorabies virus extracellular virions.
J Virol 2011, 85:6427-6441. PubMed Abstract | Publisher Full Text
Zhu FX, Chong JM, Wu L, Yuan Y: Virion proteins of Kaposi's sarcoma-associated herpesvirus.
J Virol 2005, 79:800-811. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Loret S, Guay G, Lippe R: Comprehensive characterization of extracellular herpes simplex virus type 1 virions.
J Virol 2008, 82:8605-8618. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
van Nieuwstadt AP, Dijkstra SG, Haenen OL: Persistence of herpesvirus of eel Herpesvirus anguillae in farmed European eel Anguilla anguilla.
Dis Aquat Organ 2001, 45:103-107. PubMed Abstract
Chen SN, Ueno Y, Kou GH: A cell line derived from Japanese eel (Anguilla japonica) kidney.
Gilad O, Yun S, Andree KB, Adkison MA, Zlotkin A, Bercovier H, Eldar A, Hedrick RP: Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi, Cyprinus carpio koi.
Dis Aquat Organ 2002, 48:101-108. PubMed Abstract
Ince IA, Boeren SA, van Oers MM, Vervoort JJ, Vlak JM: Proteomic analysis of Chilo iridescent virus.
Virology 2010, 405:253-258. PubMed Abstract | Publisher Full Text
Mastroleo F, Leroy B, Van Houdt R, s' Heeren C, Mergeay M, Hendrickx L, Wattiez R: Shotgun proteome analysis of Rhodospirillum rubrum S1H: integrating data from gel-free and gel-based peptides fractionation methods.
J Proteome Res 2009, 8:2530-2541. PubMed Abstract | Publisher Full Text
Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, Mann M: Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.
Mol Cell Proteomics 2005, 4:1265-1272. PubMed Abstract | Publisher Full Text
CBS Prediction Servers [http://www.cbs.dtu.dk/services/] webcite
Krogh A, Larsson B, von Heijne G, Sonnhammer EL: Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.
J Mol Biol 2001, 305:567-580. PubMed Abstract | Publisher Full Text
Emanuelsson O, Brunak S, von Heijne G, Nielsen H: Locating proteins in the cell using TargetP, SignalP and related tools.
Nat Protoc 2007, 2:953-971. PubMed Abstract | Publisher Full Text
Gupta R, Jung E, Brunak S: Prediction of N-glycosylation sites in human proteins. [http://www.cbs.dtu.dk/services/NetNGlyc/abstract.php] webcite
Julenius K, Molgaard A, Gupta R, Brunak S: Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites.
Glycobiology 2005, 15:153-164. PubMed Abstract | Publisher Full Text
Gibson W, Roizman B: Proteins specified by herpes simplex virus. 8. Characterization and composition of multiple capsid forms of subtypes 1 and 2.
J Virol 1972, 10:1044-1052. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Perdue ML, Cohen JC, Kemp MC, Randall CC, O'Callaghan DJ: Characterization of three species of nucleocapsids of equine herpesvirus type-1 (EHV-1).
Virology 1975, 64:187-204. PubMed Abstract | Publisher Full Text
Perdue ML, Cohen JC, Randall CC, O'Callaghan DJ: Biochemical studies of the maturation of herpesvirus nucleocapsid species.
Virology 1976, 74:194-208. PubMed Abstract | Publisher Full Text
Mettenleiter TC, Klupp BG, Granzow H: Herpesvirus assembly: an update.
Virus Res 2009, 143:222-234. PubMed Abstract | Publisher Full Text
Newcomb WW, Trus BL, Booy FP, Steven AC, Wall JS, Brown JC: Structure of the herpes simplex virus capsid. Molecular composition of the pentons and the triplexes.
J Mol Biol 1993, 232:499-511. PubMed Abstract | Publisher Full Text
Rosenkranz D, Klupp BG, Teifke JP, Granzow H, Fichtner D, Mettenleiter TC, Fuchs W: Identification of envelope protein pORF81 of koi herpesvirus.
J Gen Virol 2008, 89:896-900. PubMed Abstract | Publisher Full Text
Muller A, Markussen T, Drablos F, Gjoen T, Jorgensen TO, Solem ST, Mjaaland S: Structural and functional analysis of the hemagglutinin-esterase of infectious salmon anaemia virus.
Virus Res 2010, 151:131-141. PubMed Abstract | Publisher Full Text
Schutze H, Ulferts R, Schelle B, Bayer S, Granzow H, Hoffmann B, Mettenleiter TC, Ziebuhr J: Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.
J Virol 2006, 80:11598-11609. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Mocarski ES: Comparative analysis of herpesvirus-common proteins. In Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Edited by Arvin A, Campadelli-Fiume G, Mocarski E, Moore PS, Roizman B, Whitley R, Yamanishi K. Cambridge: Cambridge University Press; 2007.
Guo H, Shen S, Wang L, Deng H: Role of tegument proteins in herpesvirus assembly and egress.
Protein Cell 2010, 1:987-998. PubMed Abstract | Publisher Full Text
Makarova KS, Aravind L, Koonin EV: A novel superfamily of predicted cysteine proteases from eukaryotes, viruses and Chlamydia pneumoniae.
Trends Biochem Sci 2000, 25:50-52. PubMed Abstract | Publisher Full Text
Newcomb WW, Brown JC: Structure and capsid association of the herpesvirus large tegument protein UL36.
J Virol 2010, 84:9408-9414. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Davison AJ: Comparative analysis of the genomes. In Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Edited by Arvin A, Campadelli-Fiume G, Mocarski E, Moore PS, Roizman B, Whitley R, Yamanishi K. Cambridge: Cambridge University Press; 2007.
McGeoch DJ, Rixon FJ, Davison AJ: Topics in herpesvirus genomics and evolution.
Virus Res 2006, 117:90-104. PubMed Abstract | Publisher Full Text


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Running Windows (or anything else) on your Mac Discussion of Classic or running Windows, Linux and other OSes on the Mac. |
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I posted an earlier question about Boot Camp and fortunately got the help to fix it. Well now I went in to set up bootcamp. It rebooted my computer then to start a install of Windows XP Pro w/ SP2. Of course it asked to format the drive and I did do that with NTFS and made sure it was the new partition I created which was 28 gigs. Well for some reason during this my screen went black like my computer was not on. I had it plugged in so unless something was loose I'm sure the power didn't go dead.
Well now when I turn it on it gets to a grey screen with no writing, then a black screen, and simply says "Press any key to boot from CD" and I don't press anything and then it says "Missing operating system" I have tried pressing a key as well with no luck. The thing is even with the CD eject button I'm unable to remove the CD (windows xp) which is in the computer and what scares me is I can't even get into the Mac side. I'm completely locked out and have no idea what to do. Any suggestions? Would boot camp have somehow wiped my entire hard drive including the mac side? |
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So I ordered a genuine Windows XP install disk with SP2 and successfully got Boot Camp to run on my Macbook at the beginning of last summer. It was great. Everything ran smoothly, but yesterday, while on Windows XP, the keyboard stopped working. Not a keyboard hooked up to the Macbook via USB, but the keboard that is on the Macbook. So I was like WTF? I then restarted, booted to Windows, got to the login screen, and I couldn't type a darn thing. Even when I press the caps lock button (there's supposed to be a green light on the key) but there was none! Then I rashly decided to "uninstall" the windows partition. So I booted on Mac OSX, ran Boot Camp Assistant, partitioned the harddrives into one, then restarted a bunch of times. I then ran Software Update and updated all my firmware, so now I was ready to reinstall Windows XP on my Macbook, again. I pop in the disc and let it load. I waited until the blue screen told me to press Enter to continue, press r to repair, or press F3 to quit. I pressed Enter and nothing happened. I then pressed F3, nothing happened! Now when I turn on my Macbook, I go to the black screen which says "No bootable device--insert boot disk and press any key." From my boot camp experience, I have to hold the alt key right after pressing the power button to load Mac OSX. It's greatly appreciated if anyone has any suggestions to fix these problemS.
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Then put in the Apple disc you got with the machine and hold down C for a while until the apple logo and the little wheel thing appears. Then wait for the recovery to start, Select your language. The do nothing until the bar at the top appears. Go to Utilies then start up disc. Select the Mac OS X 10.4.8 or whatever version you are on. You will be prompted to reboot. and then it back to normal. I hope that is clear and helps and it works for me, it takes less than 5 minutes. |
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I currently have this problem. The usual suspects don't help however, i.e., holding C or option while trying to boot. In fact, when trying option key booting, it no longer shows my mac volume (or a windows volume either) )o: Cursed boot camp!!! I've pretty much resigned myself to losing data <sobs at the thought of the last few weeks of phd work down the drain>. However, I'll be happy if I can just get my iMac to boot, reinstall leopard or just plain work at all! I'm stuck at the dreaded grey screen. I'm running leopard on an intel iMac. Has anyone got any ideas? Has anyone tried booting from another mac under these circumstances with any success? Thanks in advance.
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I'm having the same problem where my unibody macbook snow leopard can't even find the os x partition after running boot camp to install xp. I was able to boot from the osx install cd but it is prompting me to startup as if it is a completely new fresh install. Is there anyway to figure out if my old partition is still there so i don't lose all my data?
More info: I initially completed the windows install with boot camp but i didn't partition with enough disk space (5GB) and ran into trouble when trying to install sp2. I repartitioned with more (40GB) after removing the initial and was never able to complete the install for windows (just kept freezing up during the install). Now when i restart it only shows the windows partition that isn't even working and it doesn't show the os x partition. Any help is greatly appreciated. |
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I have a mac pro with RAID, so I figured, I can´t have windows on my mac.
I wanted to try to install windows on an external drive (3,5" using the esata port from newer technology. Didn´t work either. I tried to install windows on that disc using parallels on my macbook. That worked, but couldn´t boot from the macpro. I installed OSX on that disc, while connected to macpro via esata in order to install windows from there using bootcamp. Didn´t work. Then I took all my 4 RAID discs out of the macpro and inserted the external which had OSX installed. After start up it came with that message: No Bootable Device -- Insert boot disk and press any key Then I changed back to my 4 RAID discs and it says the same. I tried to start while pressing the option key. Same. I also tried starting holding the T down to see if I can get access to my data, but still the same. I cannot do anything. Anybody any idea? I´m a bit desperate.... |
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The songs on White Hat range from gun-in-her-garter saloon narratives and paranoid soul struts to intimate ballads of quiet resignation and open-hearted love songs. The recordings are raw and un-fussed over, with the spirited energy and honest emotion of live performance. Drawing from the classic country and folk of Senseney's childhood and anchored by Drootin's rock-solid rhythms and indie-punk roots, White Hat contains echoes of artists from Townes Van Zandt to Nick Cave, Willie Nelson to Captain Beefheart. Fitting for a couple in no rush to slow down, White Hat was recorded at LA's Nightingale studios in a mere three days. Rilo Kiley bassist Pierre de Reeder recorded the album and drums were handled by Chris Phillips.
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Ocean Policy Update |
Number 5 – March 2006 |
Issue Highlights:WA Ocean Policy Work Group updateReport on Meeting #6 - February 28, 2006State Ocean Policy NewsOR - Next OPAC meeting on March 17 in Tilamoook, ORCA - Announcement of Ocean Protecction Council Executive Ocean Policy Officer positionFL - Latest Ocean and Coastal Resources Council meetingOther NewsSenate Subcommittee to hold Offshore Aquaculture Hearing April 6______________________________________________
Washington State
Ocean Policy Work Group Update
The Washington Ocean Policy Work Group held its second meeting of 2006 in Olympia, WA on February 28. At this meeting, student researchers from the University of Washington's School of Marine Affairs presented their draft policy research memos to the work group. Included in the meeting report are the slides from student presentations, as well as a summary of the meeting. The work group's next meeting is scheduled for Friday, March 24th from 9am to 12pm in Olympia. The main topic for this meeting will be continued planning for a pubilc outreach program, to commence in May. State Ocean
Governance Update
Note: We hope to track the most active States in ocean policy. We are currently tracking numerous State ocean policy developments. The following are selected examples, and we hope to continue to include further examples in future issues. If you have specific updates on State activities, please contact the newsletter (see left). Oregon
The next Oregon OPAC meeting is scheduled for March 17th, in Tilamook, OR. The public is invited to attend. Among the topics that will be discussed are proposed planning for an offshore national marine sanctuary, coastal seafloor mapping, and gaining public comments on the OPAC's work. View Governor Kulongoski's request to have the Oregon Coast named a National Marine Sanctuary. California Florida
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Exhibition or unclassified race Friday, August 19, 1994 at Portland Speedway, Portland, OR 20 laps on a .500 mile paved track (10.0 miles)
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Scrabble word: CHEEK
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Definitions of CHEEK in dictionaries:
- noun - either side of the face below the eyes
- noun - an impudent statement
- noun - either of the two large fleshy masses of muscular tissue that form the human rump
- noun - impudent aggressiveness
- verb - speak impudently to
- The fleshy part of either side of the face below the eye and between the nose and ear.
- Something resembling the cheek in shape or position.
- Either of the buttocks.
- To speak impudently to.
- verb - to speak impudently to
There are 5 letters in CHEEK: C E E H K
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Entourage: Season 2, Episode 14The Abyss (4 Sep. 2005) 8.6
When Ari and E learn about each-others problems with Terence viz. Vince, both of which make the press, panic is total. Drama and Turtle give Vince the cold shoulder, hoping Saigon and his ... See full summary » Director:Julian Farino |
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Locks46+91+38&35 November 16, 2012 6:35 PM WSBK » Frustrating start for Honda signing Haslam @Vilas You usually make some very good points and i tend to agree with you a lot of the time . But on this occasion i was one of those who hit the disagree button . Yes Rea does send it down the road a lot but same as has been mentioned many times . You only need to look at the Level of his team mates and their performances to see how close to the edge and beyond Rea takes that bike . Hopefully with Honda on board they might be able to even things up a bit . Reply Report abuse 2 agree 0 disagree
Locks46+91+38&35 November 16, 2012 6:25 PM WSBK » Frustrating start for Honda signing Haslam @conger And you have the nerve to call someone like Haslam a numpty . I can imagine what sort of level your feedback to a race engineer would be . People like you and those who agreed with you know nothing about what's required to be a World ranked racer . Reply Report abuse 6 agree 2 disagree
Locks46+91+38&35 November 16, 2012 6:04 PM MotoGP » Colin Edwards eyeing FTR-M1 for 2014? @Idiocypersonified Hey man you know what ! I couldn't of put it better myself . So there is nothing to add . Just that i'm in total agreement . Reply Report abuse 1 agree 0 disagree
Locks46+91+38&35 November 13, 2012 5:58 PM MotoGP » Valencia MotoGP test times - Tuesday (Final) @ Nicky still beating Rossi . How pathetic some people are . Reply Report abuse 15 agree 4 disagree
Locks46+91+38&35 November 13, 2012 5:35 PM MotoGP » Rossi, Lorenzo abandon Valencia MotoGP test How the hell has this become a thread about Casey Stoner . The attention span of some people seems to be non existant . Reply Report abuse 12 agree 0 disagree
Locks46+91+38&35 November 11, 2012 4:36 PM MotoGP » Moto2: Marquez storms through the field for Valencia win What is the relevance of the Dani and Cal posts . Moto GP is going to be very interesting from testing on Tuesday right through to the final race of next season . Reply Report abuse 1 agree 0 disagree
Locks46+91+38&35 November 09, 2012 3:15 PM MotoGP » Rossi: 'Emotional' Sunday, 'can't wait' for Tuesday One thing that comes to mind reading all the anti Rossi comments is " What the hell is Hayden doing in Moto GP then " . Reply Report abuse 0 agree 0 disagree
Locks46+91+38&35 November 07, 2012 5:11 PM F1 » Raikkonen: I'm not stupid! @Brian Conn If you actually listen to the interview he tells you the whole story but in very few words . And all this fuss about Kimi and yet i don't see too much been said about Vettel who was far worst by using the F word . I don't see a problem with Kimi maybe Vettel was a bit past the mark but i still wouldn't crucify him for it . I once heard a mother telling someone off for swearing infront of her effing kids . Yes she used that word . Reply Report abuse 2 agree 0 disagree
Locks46+91+38&35 November 07, 2012 12:34 AM Last Edited 911 days ago F1 » Raikkonen: I'm not stupid! Damn ! Oooopss hope the puritan gets too offended to read this far down the list . What planet is that person on . Kimi is absolute gold . And Seb well he just proved he is human and i respect him for been as close to normal as you can be in that enviroment.David Coulthard should go and get a role in the Archers if he feels he is above that. As for The Puritan i think you got the message . I would hate the thought of spending the shortest amount of time in your company . Or the company of the few that agreed with you . Loosen up man it could all end tomorrow . Reply Report abuse 6 agree 1 disagree
Locks46+91+38&35 November 06, 2012 4:00 PM MotoGP » Valentino Rossi: 'It will be a strange weekend' @Dave Smithers So you are saying that if you were racing at the speeds these guys do and you got on a bike that was the opposite to what your riding style requires you really would just go out there and ride it past the point you know you might crash .The guy isn't afraid to race he just sees it as a risk he would rather not take . And i respect him for been honest instead of throwing himself down the road like Hayden has done . The bench mark is the number of times he has finished ahead of Hayden . And even Hayden says the bike has got better . Also Casey won less and less as the seasons went on . So that alone shows how far behind the bike had got because Ducati assumed if casey could go fast on it then there wasn't a problem .Casey has a unique style that suited the bike but even he decided enough is enough and moved on . I don't think he moved just for money because Casey isn't motivated like that. Anyway i will take this oppurtunity to say HE WILL BE MISSED . Reply Report abuse 15 agree 2 disagree


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Emraan Hashmi is now being compared to Salman Khan! Reason: Like Salman Khan, almost all Emraan Hashmi films have become a hit at the box office.
Emraan feels honored to be compared to the star.
"I hope my films keep becoming hits, and being compared to Salman is a huge honour," the 33-year-old said.
"I think I have got a lot of work to do, I have been in the industry for just nine years. He is a senior and very encouraging," added Emraan.
Emraan Hashmi's latest movie Raaz 3, which released on Friday, is going strong at the BO.
Wondered why the actor has always featured in bold films only??
Well, Emraan said: "No one gives me such (universal) films. When someone gives me a universal subject, I'll do it."
Emraan's forthcoming films include Ghanchakkar , Ek Thi Daayan and Unglee.
Follow Bollywood Celebden on Facebook and stay updated with bollywood news throughout the day!
2 thoughts on "Why is Emraan Hashmi being compared to Salman Khan??"
Haha… What a joke.. Emraan was compared with a tiger… Hahaha.
Emraan is younger than Salman.


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Petascale computing
This article is outdated. (April 2014) |
This article includes a list of references, but its sources remain unclear because it has insufficient inline citations. (February 2009) |
In computing, petascale refers to a computer system capable of reaching performance in excess of one petaflops, i.e. one quadrillion floating point operations per second. The standard benchmark tool is LINPACK and Top500.org is the organization which tracks the fastest supercomputers. Some uniquely specialized petascale computers do not rank on the Top500 list since they cannot run LINPACK. This makes comparisons to ordinary supercomputers hard.
Petascale can also refer to very large storage systems where the capacity exceeds one petabyte (PB).
Petascale computing is being used to do advanced computations in fields such as weather and climate simulation, nuclear simulations, cosmology, quantum chemistry, lower-level organism brain simulation, and fusion science.
The National Science Foundation is responsible for initiating and funding several petascale computers in the USA, as well as DARPA who gave IBM the contract to develop the petascale PERCS (Productive, Easy-to-use, Reliable Computer System) platform.
China has developed three petascale computers, Nebulae, Tianhe-I and Tianhe-2.
Russia has developed the Lomonosov (rus) petascale computer.
Other countries, such as Germany and Japan, have plans of their own for petascale computers.
Petascale computers are under development from manufacturers such as Sun Microsystems, Cray, IBM, Dawning, SGI, and NEC.
As of 2012, these are the known active petascale computers in the world.
- Roadrunner, built by IBM, was the first computer to go petascale, and did so on May 25, 2008, with sustained performance of 1.026 petaflops.
- XT5 "Jaguar", built by Cray, was the second, later in 2008. After an update in 2009, its performance reached 1.759 petaflops.[1]
- SGI Pleiades which went online in 2008 with a performance of 600 TFLOPS, reached petascale in 2012.
- Nebulae built by Dawning, was the third petascale computer and the first built by China with a performance of 1.271 petaflops in 2010.
- Tianhe-1A built by NUDT, at 2.566 petaflops in 2010.
- K computer built by Fujitsu, is the second fastest supercomputer in the world, at 8.162 petaflops in 2011.
- Tsubame built by NEC/HP
- Cielo built by Cray
- Hopper built by Cray
- Tera 100 built by Bull SA
- IBM Sequoia
- Cray Titan, an updated version of Jaguar.
The first 20 supercomputers on the June 2012 list are petascale.
- RIKEN MDGRAPE-3 in Japan which went online in 2006 reaches petascale performance but can't run LINPACK, so comparisons to regular supercomputers are hard.
Under construction[edit]
- IBM Blue Waters -- recently ended their partnership.[needs update]
- JUGENE, an upgraded Blue Gene/P system at Jülich Research Centre in Germany, operational in mid 2009.[2]
- Tianhe-2
Platform examples[edit]
- SGI Altix
- Sun Constellation System
- IBM Blue Gene/P, Blue Gene/Q and PERCS
- Cray XT5, XT6, XE6, XK6
See also[edit]
- ^ National Center for Computational Sciences (NCCS) (2010). "World's Most Powerful Supercomputer for Science!". NCCS. Retrieved 2010-06-26.
- ^ IBM Press Release (2009-02-10). "New IBM Petaflop Supercomputer at German Forschungszentrum Juelich to Be Europe's Most Powerful". IBM Inc. Retrieved 2010-06-26.


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Alabama Softball Mississippi State at #4 Alabama May 12, 2011 at OXFORD, Miss. (Ole Miss Complex)
Score by Innings R H E ----------------------------------------- Mississippi State... 001 000 0 - 1 2 1 Alabama............. 000 131 X - 5 9 1 -----------------------------------------
Umpires - HP: John O'Meilia 1B: Keith Kearney 3B: David Erwin Start: 6:35 PM Time: 2:19 Attendance: 1080 Weather: Fair, 84, Wind: SSW at 3 p.m.
Mississippi State starters: 1/ss A Bainbridge; 22/2b H. Shape; 7/lf K. Smith; 25/1b C. Nesbit; 16/c S. Lenahan; 15/3b C. Raines; 21/rf Briana Bell; 2/dp J. Cooley; 4/cf B. Gates; 11/p S. Becker; Alabama starters: 1/lf Braud; 7/cf Fenton; 20/ss Larsen; 22/1b Locke; 33/dp Traina; 2/rf Lunceford; 10/2b Hunt; 12/c Dawson; 9/3b Conley; 0/p Dunne; Mississippi State 1st - A Bainbridge walked (3-2). H. Shape walked (3-2); A Bainbridge advanced to second. K. Smith reached on a fielder's choice (1-2); H. Shape advanced to second; A Bainbridge out at third p to 3b. Dropped foul ball, E3. C. Nesbit flied out to rf (2-2). S. Lenahan walked (3-2); K. Smith advanced to second; H. Shape advanced to third. C. Raines fouled out to 1b (0-2). 0 runs, 0 hits, 1 error, 3 LOB. Alabama 1st - Braud singled to third base, bunt (1-1). Fenton singled to pitcher, bunt (0-0); Braud advanced to second. Larsen popped up to p (2-2). Locke popped up to 2b (0-1). Traina grounded out to 1b unassisted (0-0). 0 runs, 2 hits, 0 errors, 2 LOB. Mississippi State 2nd - Briana Bell struck out swinging (1-2). J. Cooley struck out swinging (2-2). B. Gates struck out swinging (0-2). 0 runs, 0 hits, 0 errors, 0 LOB. Alabama 2nd - Lunceford out at first p to 2b, bunt (0-0). Hunt singled to center field (2-1). Dawson fouled out to c (0-2). Hunt out at first 1b unassisted, runner left early. 0 runs, 1 hit, 0 errors, 0 LOB. Mississippi State 3rd - A Bainbridge grounded out to 3b (1-1). H. Shape hit by pitch (3-1). K. Smith reached on a fielder's choice (0-2); H. Shape out at second ss to 2b. C. Nesbit walked (3-2); K. Smith advanced to second. S. Lenahan singled to center field, RBI (1-2); C. Nesbit advanced to second; K. Smith scored. C. Raines struck out swinging (3-2). 1 run, 1 hit, 0 errors, 2 LOB. Alabama 3rd - Conley popped up to 2b (3-1). Braud reached on a muffed throw by 2b, assist by 1b (1-0). Braud out at second c to ss, caught stealing. Fenton struck out swinging (3-2). 0 runs, 0 hits, 1 error, 0 LOB. Mississippi State 4th - Briana Bell grounded out to 3b (2-1). J. Cooley walked (3-1). S. Fisher pinch hit for B. Gates. S. Fisher fouled out to 1b (1-2). A Bainbridge struck out swinging (0-2). 0 runs, 0 hits, 0 errors, 1 LOB. Alabama 4th - B. Gates to cf for S. Fisher. Larsen doubled to left field (0-0). Locke lined out to 3b (1-1). Traina walked (3-1). Traina advanced to second; Larsen advanced to third on a passed ball. Lunceford flied out to lf, SF, RBI (2-2); Larsen scored, unearned. Hunt struck out swinging (1-2). 1 run, 1 hit, 0 errors, 1 LOB. Mississippi State 5th - H. Shape out at first p to 2b, bunt (2-1). K. Smith grounded out to 2b (2-1). C. Nesbit struck out swinging (0-2). 0 runs, 0 hits, 0 errors, 0 LOB. Alabama 5th - Patterson pinch hit for Dawson. Patterson struck out swinging (2-2). Conley flied out to lf (2-1). Braud singled to left center (1-1). Fenton singled to third base (1-0); Braud advanced to second. Larsen homered to left center, 3 RBI (2-2); Fenton scored; Braud scored. Locke singled to center field (2-2). Traina struck out swinging (1-2). 3 runs, 4 hits, 0 errors, 1 LOB. Mississippi State 6th - Dawson to c for Patterson. S. Lenahan lined out to ss (1-1). C. Raines struck out swinging (0-2). Briana Bell struck out looking (2-2). 0 runs, 0 hits, 0 errors, 0 LOB. Alabama 6th - Lunceford tripled to left center (2-2). Hunt flied out to rf, SF, RBI (0-0); Lunceford scored. Dawson grounded out to ss (0-0). Conley fouled out to c (1-1). 1 run, 1 hit, 0 errors, 0 LOB. Mississippi State 7th - J. Cooley struck out swinging (2-2). Boccia to 1b for Locke. B. Gates struck out swinging, out at first c to 1b (1-2). A Bainbridge singled up the middle (1-2). H. Shape popped up to p (3-2). 0 runs, 1 hit, 0 errors, 1 LOB.


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Dell has released its annual Threat Report, highlighting emerging threat trends and looking at the most common attacks observed in 2015. The report is based on research from Dell's Global Response Intelligence Defense
On the one hand, worldwide PC shipments dropped by 6.7 per cent year-over-year in the first quarter of 2015, but on the other hand that's not as bad a decline as IDC Corp.
It's International Women's Day on March 8 and to celebrate, we're celebrating women business leaders in the technology space - with a little help from OpenText, the largest Canadian-based software company. The five
PC sales are slowly yet surely creeping their way upwards, with analysts pointing to a one per cent increase in the number of PCs shipped worldwide as a clue that the market is
When he was on assignment for the FBI in its Los Angeles field office, John McClurg was charged with bringing down hacker Kevin "Dark Dante" Poulson. Poulson orchestrated a hack of Pacific Bell's
PC sales may be in for a slow rebound, thanks to more first world consumers already owning tablets and starting to buy desktops again. On Thursday, research firm Gartner Inc. reported 79.4 million
A fundraising round for a cloud-based video surveillance system has attracted an unlikely pair of investors in Michael Dell and Enrique Salem. Austin, Texas-based Eagle Eye Networks Inc. announced this morning that MSD
Good news for PC makers today – while sales of the desktop computer aren't exactly booming, they're no longer plunging the way they have been for the past two years either, according to
Samsung Electronics, Intel Corp., and Dell Inc. have banded together to create a new Internet of Things standard, ensuring there's a common framework for how devices talk to each other. Alongside chip makers
Along with the MacBook Air, the Dell XPS 13 is the only other consumer model in this list. However, Dell does offer it with Windows 8.1 Professional factory installed, and is marketing it
Dell's Latitude series of notebooks have long been their premium series of business workhorses, and for 2014, the new top of the range 7000 series are the first Ultrabook Latitudes. Dell calls them
"Sell the sizzle not the steak" is an old saying in the marketing biz, but channel partners and their customers got to the meat (figuratively and literally) of software defined networking (SDN) during


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New Delhi, Jan 30 (IANS) The cabinet committee on investment Wednesday asked the defence and petroleum and natural gas ministries to resolve in one month issues linked to the exploration activities in 39 New Exploration Licensing Policy oil (NELP) and gas blocks.
The meeting was presided over by Prime Minister Manmohan Singh.
An official release said: "The issues of the ministry of defence and ministry of petroleum and natural Gas were discussed in detail. The CCI has directed that both the defence ministry and the petroleum ministry discuss the issue in detail and formulate solutions within one month."
Information and Broadcasting Minister Manish Tewari said the oil and natural gas sector was discussed during the meeting.
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As the KVNF Fall Pledge Drive wraps up, we want to again express our sincere thanks and gratitude for all your support of this station. It's been an incredibly heartwarming week talking to all of you and hearing why you value community radio. This is your community radio station, and you've all shown it this week with your generous contributions.
Your donations go directly into our operating expenses and paying for the quality programming you hear every day.
It's here! The KVNF Fall Pledge Drive Kicked off this morning and runs through Saturday, October 12th.
Pledge Drives are hugely important to community radio stations like us - your support makes up our largest source of funding. To put it simply - we wouldn't be here without you! Your donations go directly to everything from daily operating expenses to paying for the informative and entertaining programming you hear every day.
Can you help?
Two NFCB (National Federation of Community Broadcasters) member radio stations suffered extreme damage during hurricane Sandy. Visit the websites of WFMU and WBAI to find out more, and see how you can help.


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At Kesteven & Grantham Girls School, we pride ourselves not only on our results but on the rounded and accomplished young women who graduate each year. We have a consistent record of academic excellence at the highest levels achieved in an environment that encourages our pupils to grow and mature into capable, confident adults. Read More
Inspire, Challenge, Achieve
01.09.2015 University Entrance Exams
KGGS is happy to accept applications from private candidates who wish to register for university entran... Read More -
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The best girls school that plays Waterpolo in the country at u16 level. ESSA U16 Waterpolo Champions! ... Read More -
02.03.2015 Concerns Expressed Over Free School Transport
Parents from Claypole and a number of other villages are campaigning to change LCC policy regarding fre... Read More -
04.02.2015 Student Finance Applications 2015/16
The application cycle for Academic Year 2015/16 opened on 26th January 2015. Applications can be made b... Read More
05.05.2015 Year 8 Head of Year Reward Evening
Year 8 Head of Year Reward Evening... Read More


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FavouriteOne to whom a lady gives a "favour" or token. The horse which betting men suppose is most likely to come off the winner of a particular race. Source: Dictionary of Phrase and Fable, E. Cobham Brewer, 1894 More on Favourite from Fact Monster:
24 X 7Private Tutor
Explore Online Physics Tutor , Math Answer


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use the following search parameters to narrow your results:
e.g. subreddit:aww site:imgur.com dog
subreddit:aww site:imgur.com dog
see the search faq for details.
advanced search: by author, subreddit...
~9 users here now
Need help? You're not alone. If you are suicidal, call 08457 90 90 90 in the UK, or 1800 273 8255 in the US. In Australia, visit lifeline, for phone numbers and online chat.
7 Cups of tea is an emotional support service, connecting people who are sad, in distress, lonely, or in pain, with trained active listeners. We are helping over 11,000 people every week from over 80 countries.
Alternatively, if you want to keep it in the family, there are plenty of good people you can talk to at /r/suicidewatch.
If you have stumbled upon /r/sad by accident and have become depressed as a result, visit /r/happy, /r/kittens, /r/upliftingnews or /r/trees.
If you're...sad, depressed or just need a hug, you've come to the right subreddit. This is a place where you can share your own stories and help others with theirs. Feel free to also post anything and everything you find sad.
Just a few ground rules: Never EVER make fun of anyone's story. It takes a lot of bravery to post here. If you have just come here to laugh at other peoples' misfortune, please do so to yourself. Everyone is different and is saddened by different things, so please don't go freaking out on someone because something they found sad wasn't sad to you.
Also, take a look at these subreddits: r/foreveralone r/lovehurts r/sadcomics /r/FunnyandSad /r/PsychWard
I'm normal (self.sad)
submitted 2 years ago by demonslay3r
It gets annoying when people say "oh your so cold hearted, you are mean ETC." Just because someone who was locally famous for something dies or anyone. I've seen over 23 of my friends die. I'm used to death. If your not in anyway connected out affiliated with me. My mind just tells me "that's not your problem man its just another useless thing " I agree with my mind after that.I actually don't feel sorry for you because your in heaven. Its better then living Down here, for all you know you could be getting robbed and about to get killed while reading this. In heaven there is no worries about anything. Now stop saying I have no heart and most of you redditors will have to deal with it.
there doesn't seem to be anything here
π Rendered by PID 1104 on app-04 at 2015-04-19 01:30:57.157982+00:00 running 301cfbe country code: US.


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Title: | Native Species Regeneration Following Ungulate Exclusion and Nonnative Grass Removal in a Remnant Hawaiian Dry Forest. |
Author: | Thaxton, Jarrod M.; Cole, T. Colleen; Cordell, Susan; Cabin, Robert J.; Sandquist, Darren R.; Litton, Creighton M. |
Date: | 2010-10 |
Publisher: | Honolulu, University of Hawaii Press |
Citation: | Thaxton JM, Cole TC, Cordell S, Cabin RJ, Sandquist DR, Litton CM. Native Species Regeneration Following Ungulate Exclusion and Nonnative Grass Removal in a Remnant Hawaiian Dry Forest. Pac Sci 64(4): 533-544. |
Abstract: | Hawaiian lowland dry forests have been reduced by >90% since first human contact. Restoration has focused on protection from fire and ungulates, and removal of invasive grasses as ways to stimulate native forest regeneration. Despite these efforts, natural regeneration of native plants has been infrequent. To assess effects of previous restoration treatments on natural regeneration, we monitored seed rain and dynamics of seedlings and juveniles for a period of 3 yr (2004–2007) within three restoration units within a remnant forest on the island of Hawai'i. All units had been protected from fire for many decades but differed in time since ungulate exclusion and grass removal. The units were as follows: (1) long-term restoration (fenced 1956, grass removal initiated 1995), (2) shortterm restoration (fenced and grass removal initiated 1997), and (3) unmanaged (fenced 1997, no grass removal). Overall juvenile plant abundance was highest in the short-term unit, but native abundance was highest in the long-term unit. Native woody seedlings established in all units, but recruitment into larger size classes was restricted to units with grass removal, primarily the long-term unit. Native seed rain explained much of the variation in native seedling abundance between units with grass removal. Nonnative grass seed rain was extensive but was reduced by an order of magnitude with grass removal. This study suggests that natural regeneration may enhance restoration actions in sites with native canopy, but this is likely only when restoration activities have been maintained for several years to coincide with favorable rainfall conditions that are highly unpredictable over time. |
Series/Report No.: | vol. 64, no. 4 |
Description: | v. ill. 23 cm.
Quarterly |
Pages/Duration: | 12 p. |
URI: | http://hdl.handle.net/10125/23124 |
LC Subject Headings: | Natural history--Periodicals. Science--Periodicals Natural history--Pacific Area--Periodicals. |
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vol64n4-533-544.pdf | 624.2Kb |
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[Date Index] [Thread Index] [Author Index]
nice Gotcha!
Code similar to the following gave me trouble today. I thought it would be fun to share: Clear[x]; test[] := Module[ {x}, ToExpression["x=3"]; Print[x]; ]; test[]; Print[x]; In my version. "x" was infact the variable "Active" which is an option to ButtonBox and created a ton of problems. Of course I could not change the name (user driven, and that's why I am running a ToExpression on it). The solution was to not declare Active in the Module and to Clear[Active] in the function. any other insight? Luc Barthelet GM the day, Mayor at night http://www.simcity.com


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Hello everyone! I recently purchased a Bushmaster online to use as my patrol rifle. I was originally going to run an eotech alone on it but later found out that it's policy to have iron sights on the gun at all times. Right now I have the magpul moe hand guards on and I need to figure out what I can do on the front sight and I have no clue! I want the magpul rear flip up sight, then my eotech and a flip up sight on the front. Can someone tell me how I can make this happen easily with what I have on it right now? The first photo is the before picture how I bought it
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this is the current pic of the rifle
Change your gas block out to a front sight. Or one with a rail to add a collapsible site.
install 1 of these - http://www.midwestindustriesinc.com/index.cfm?fuseaction=category.display&category_ID= 252 and get yourself a set of these - http://www.midwestindustriesinc.com/index.cfm?fuseaction=category.display&category_ID= 362
Member: NRA GOA ΜΟΛΩΝ ΛΑΒΕ Est sularas oth mithas "either way, you were guilty by association, so you were smited...." JD
Hate to see you lose your hand guard, but replacing it with a quad rail may be another option.
Magpul make a flip up front that fits the moe hand guard or the gas block and it will co-witness.


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Online Pooja | Homams
The existence of a super natural power that controls all goings-on in the universe is a belief that exists from time immemorial in all geographical areas ,in all tribes , religions, sects, castes and creeds. The name and mode of worship of that unknown power varies but the basic notion or concept of the God is same. In Hinduism the faithful worship God in many forms but it rely on one God. "UPASAKANAAM KARYARTHAM BRAHMANO ROOPA KALPANA" is the principle behind it.
Ancient saints (Rshis)had prescribed many rituals like japa, pooja,parayana, homa etc for the spiritual as well as physical wellbeing of the mankind. Hindu scriptures contain hundreds of such rituals. Our ancestors have been doing these rituals for thousands of years and these rituals like homam, pooja etc are relevant even in this space age because these have fair degree of certainty in giving positive results and many have benefitted from it.
Many people who seek divine blessings for success in their endeavours or celestial intervention to end or forestall difficulties intend to perform pooja, homam etc but are ignorant about the types of ritual they need to do or who would do it strictly in accordance with the tenets of holy scriptures. ......................................................... seeks to solve this predicament by giving brief description of common homams and poojas with it's main objectives. We have also enlisted a panel of eminent Purohits (Priests) who are scholars in Vedic doctrines and well versed in Aagama rituals to perform these homams and poojas . These Purohits are from traditional Brahmin families who have been in priesthood at the legendary MOOKAMBIKA TEMPLE of KOLLUR. This ensures authenticity of the ritual.
The list of Homams and Poojas and it's objectives given herein are not exhaustive. There are many other potent Homam that the aspirants can perform to fulfil various objective. Many of the Homams can be performed on different scale or magnitude. The uninformed can consult the Purohits on our panel free of cost to elicit more information about pooja and homam.
Though the homam can be performed at any serene and virtuous place, the effect is more pronounced when it is done at a sacred place. These places have the natural positive phenomena and that is why people go to holy places to perform religious rituals. KOLLUR the abode MOOKAMBIKA is an unmatched sacred place where we arrange to perform the homams. Kollur Mookambika Temple has been described as a world renowned "SIDDHI KSHETRA" meaning seat of mystic power, in ancient holy scriptures and as such is a very ideal location for spiritual rituals.


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6-String Nylon Acoustic-Electric Guitar w/ Gigbag
Product Code 65660
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The Multiac Encore is equipped with custom electronics by EPM. This easy to use Blendable Dual Source System features an undersaddle transducer as well as an acoustic soundboard transducer, with the ability to blend one into the other. Ideal guitar for players looking for a an incredible, amplified nylon string sound and feel in a live and loud band setting with virtually no feedback.
The Multiac Encore nylon string, also features a 25 1/2" scale and a comfortable nut width, making it easy for electric steel string players to transition to nylon strings. This model features a chambered silver leaf maple body with a solid cedar top, as well as a dual source preamp with volume, treble, mid, bass and blend control sliders.
* Scale Length: 25 1/2" * Nut Width: 1 7/8" * Electronics: Blendable Custom Dual source electronics with under-saddle transducer & acoustic soundboard transducer.
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- Neck: Mahogany
- Body: Silver Leaf Maple
- Top: Solid Cedar
- Fingerboard: Rosewood
- Fingerboard Radius: 16"
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