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Nanoparticle exposure reactivates latent herpesvirus and restores a signature of acute infection
- Christine Sattler1,
- Franco Moritz2,
- Shanze Chen1,
- Beatrix Steer3, 4, 5,
- David Kutschke1,
- Martin Irmler6,
- Johannes Beckers6, 7, 8,
- Oliver Eickelberg1,
- Philippe Schmitt-Kopplin2,
- Heiko Adler†3, 4, 5Email author and
- Tobias Stoeger†1Email authorView ORCID ID profile
DOI: 10.1186/s12989-016-0181-1
© The Author(s). 2017
Received: 24 June 2016
Accepted: 15 December 2016
Published: 10 January 2017
Inhalation of environmental (nano) particles (NP) as well as persistent herpesvirus-infection are potentially associated with chronic lung disease and as both are omnipresent in human society a coincidence of these two factors is highly likely. We hypothesized that NP-exposure of persistently herpesvirus-infected cells as a second hit might disrupt immune control of viral latency, provoke reactivation of latent virus and eventually lead to an inflammatory response and tissue damage.
To test this hypothesis, we applied different NP to cells or mice latently infected with murine gammaherpesvirus 68 (MHV-68) which provides a small animal model for the study of gammaherpesvirus-pathogenesis in vitro and in vivo. In vitro, NP-exposure induced expression of the typically lytic viral gene ORF50 and production of lytic virus. In vivo, lytic viral proteins in the lung increased after intratracheal instillation with NP and elevated expression of the viral gene ORF50 could be detected in cells from bronchoalveolar lavage. Gene expression and metabolome analysis of whole lung tissue revealed patterns with striking similarities to acute infection. Likewise, NP-exposure of human cells latently infected with Epstein-Barr-Virus also induced virus production.
Our results indicate that NP-exposure of persistently herpesvirus-infected cells – murine or human – restores molecular signatures found in acute virus infection, boosts production of lytic viral proteins, and induces an inflammatory response in the lung – a combination which might finally result in tissue damage and pathological alterations.
KeywordsCarbonaceous nanoparticles (CNP) Double-walled carbon nanotubes (DWCNT) Intratracheal instillation Persistent virus infection Virus reactivation Phospholipids
The rapid expansion of nanotechnology is expected to bring considerable benefit to mankind, but at the same time, newly developed materials might pose new and unknown risks to exposed people. Inhalation of high levels of spherical carbonaceous nanoparticles (CNP) – surrogates for combustion derived nanoparticles – has been shown to induce an inflammatory phenotype in the lungs of healthy mice [1] as well as acute extra-pulmonary cardiovascular distress [2]. Comparing a panel of different CNPs revealed particle surface related oxidative stress to be the common driver of the acute response to particles of low solubility and low toxicity [3, 4]. At moderate doses, CNP-triggered acute inflammation has been shown to resolve within several days after exposure in noncompromised mice [5]. Yet, repeated inflammatory events or exposure of individuals with higher susceptibility to NP-associated adverse effects, such as asthmatics [6], might provoke severe damage to the lung tissue. Recent research indicates that at equal surface dose, fiber shaped carbon nanotubes (CNT) – which due to their rapidly increasing mass production gain increasingly more environmental importance – also generate an acute inflammatory response via oxidative stress pathways, but in contrast to spherical particles, the CNT-induced pulmonary inflammation persists over weeks [7]. Inhaled nanoparticles generally deposit efficiently and persistent in the respiratory tract, and may due to their pro-inflammatory potency represent one environmental factor contributing to the development of lung diseases including asthma, chronic obstructive lung disease (COPD) and potentially even interstitial pulmonary fibrosis or cancer [8]. An additional environmental factor driving the susceptibility for chronic lung disease could be virus infection. A number of studies imply that especially herpesviruses might contribute to the development of lung diseases such as idiopathic pulmonary fibrosis (IPF). In lungs of patients affected from IPF, DNA of one, two or even more herpesviruses has been detected by PCR, suggesting an association between chronic virus infection and IPF [9, 10]. In particular, proteins and DNA from Human Cytomegalovirus (HCMV) and Epstein-Barr-Virus (EBV) have frequently been detected. Due to the species-specificity of HCMV and EBV, pathogenic studies of the human infections are restricted. Thus, animal models are needed and the murine gammaherpesvirus 68 (MHV-68) provides such an animal model [11, 12]. MHV-68 has been shown to act as a cofactor in bleomycin-induced fibrosis [13, 14] and to exacerbate fluorescein isothiocyanate-induced pulmonary fibrosis [14, 15]. Furthermore, infection of Th2-biased mice with MHV-68 induces the development of progressive lung fibrosis with pathological features also seen in IPF [16]. Upregulation of profibrotic or proinflammatory factors in infected cells and repeated virus reactivation followed by lytic replication events are supposed to be important factors in the development or exacerbation of IPF in these models [17, 18]. Control of viral latency and prevention of productive virus replication depends on a highly complex balance between immune surveillance and regulation of viral and cellular gene expression [12, 19, 20]. Both the immune response and the metabolism are important players in surveillance of viral latency and regulation of immune responses within this context. Viruses have been shown to alter metabolic pathways of their host cells in a highly specific manner to generate optimal conditions not only for virus replication and production of new virus particles but also for maintenance of viral latency [21, 22]. Simultaneously, immune responses and metabolism are increasingly considered to be closely linked, e.g. by sharing pathways and being crossregulated [23, 24], suggesting a well-established balance between metabolic alterations caused by the virus and modifications due to counteractions by the host. We hypothesize that inhalation of NP as a second hit disrupts this balance and interferes with the ability to control viral infection, which might finally result in a non-resolving, chronic inflammation and even fibrosis. At present, no information about the health relevance of a suchlike liaison of NP and persistent virus infection is available.
In this study, we show that the presence of NP induces the production of lytic virus from persistently infected murine cells in vitro. In vivo, exposure to NP by intratracheal instillation leads to an increase in the expression of lytic viral proteins in lungs of mice persistently infected with MHV-68 and creates transcriptome and metabolome signatures in the lung with considerable parallels to the ones observed during the acute phase of virus infection. NP exposure of human cells that are latently infected with EBV also induces reactivation of latent virus, indicating that the NP-effect is not limited to the murine system. Taken together, our results suggest that the combination of persistent herpesvirus infection and NP exposure disrupts the immune control of viral latency by altering cellular metabolism and gene expression.
NP exposure in vitro boosts lytic virus replication in pseudo-latently infected cells
NP exposure of persistently infected cells reactivates latent virus
In summary, our in vitro results show that exposure of persistently infected cell lines to carbonaceous NP induces virus reactivation and alters the expression of cellular genes associated with virus reactivation independently of the NP's type or aspect ratio, whereas acute virus growth in cells after de novo infection is not affected.
Treatment of latently infected mice with NP induces the expression of lytic virus proteins in the lung
Short-time treatment of latently infected mice with NP creates a transcriptome signature with parallels to acute virus infection
Next, we investigated if there is a match in the lung transcriptome between acutely infected mice and latently infected mice treated with NP. For that reason, total RNA from lung samples was isolated, processed and analyzed with the Illumina-MouseRef-8v2.0 Expression BeadChip as described in Materials and Methods. We scanned the data for genes that were both regulated in the acute infection situation and in latent infection plus NP compared to untreated mice and mice with latent virus alone. In the group "latent infection plus CNP", we found an overlap between this group and the group of acutely infected mice of 208 significantly altered genes (116 upregulated and 92 downregulated) from which 34 genes were at least 1.5-fold changed compared to the control groups (30 upregulated, 4 downregulated; Fig. 5e). Some of the induced genes (such as Saa3, Timp1, Cxcl1, Slc26a4, Lcn2, Ch25h and Cd14) have recently been described to be also induced by instillation of a single high dose of 162 μg CNP in the lungs of mice [40]. 18 genes including the aforementioned showed upregulation in mice with CNP only in our experiments as well, even after exposure to the comparatively moderate dose of 50 μg (Additional file 1: Table S1). As differential expression of these genes is also detected during acute virus infection, they apparently are representatives of general pathways of inflammation and immune response that are both induced during acute infection and other inflammatory events. To test if the differentially expressed genes indeed represented specific cellular pathways, data were analyzed by the use of Ingenuity Pathway Analysis (IPA, https://www.ingenuity.com). As expected, functional pathways found to be differentially regulated in latently infected mice treated with CNP as well as in acutely infected mice involved immune functions such as "activation of leukocytes" or "invasion of cells", but also pathways associated with cell proliferation (Fig. 5g). In the group "latent infection plus DWCNT", the overlap with the group of acutely infected mice consisted of 369 significantly altered genes (156 upregulated, 213 downregulated) and expression of 49 of these genes was at least 1.5-fold changed compared to the control groups (43 upregulated; 6 downregulated; Fig. 5f). Similarly to the results observed in mice treated with CNP, 19 of these 49 genes were also regulated by DWCNT alone (Additional file 1: Table S1). Data analysis using IPA showed an increase in inflammatory pathways and a reduction in "import of D-glucose" (Fig. 5h).
Additionally, we analyzed the transcriptome data by an unbiased approach for differential regulation of genes after combination of latent virus and particle exposure compared to all control groups, and heatmaps showing these genes were generated (Additional file 1: Fig. S5). In the group treated with virus and CNP, 257 differentially regulated genes were detected (110 upregulated, 147 downregulated) and 38 of these genes showed at least 1.5-fold changed expression values compared to all control groups (35 upregulated, 3 downregulated; Additional file 1: Fig. S5a). Data analysis using IPA revealed differential regulation of pathways associated with migration, activation and proliferation of cells (Additional file 1: Fig. S5c). In the group treated with virus and DWCNT, 881 differentially regulated genes were found (288 upregulated, 593 downregulated), and 197 of these genes were at least 1.5-fold changed compared to controls (92 upregulated, 105 downregulated; Additional file 1: Fig. S5b). Data analysis with IPA depicted regulation of pathways involved in proliferation and organization of cells, but also tumor-associated pathways (Additional file 1: Fig. S5d). In contrast, the IPA pathways assigned to only CNP or DWCNT (24 h) treated mice depicted mainly the inflammatory pathways: inflammatory response, cell movement, leukocyte migration and recruitment of leukocytes (data not shown). The results obtained with the Illumina expression BeadChip were validated by quantitative real-time PCR for 6 selected genes (Additional file 1: Fig. S6).
Our transcriptome data indicates that exposure of latently infected lungs to NP creates a unique expression profile which cannot be observed in untreated controls or in latently infected mice without a second hit and only to some extent in mice that were exposed to NP only. The observed transcriptome signature shows similarities to the one seen in acute virus infection and is characterized by the stimulation of inflammatory processes and the induction of an immune response.
Short-time treatment of latently infected mice creates a metabolite pattern in lung tissue with high similarity to the metabolite composition in acute virus infection
In order to illustrate the involvement of their corresponding compound classes with the found multivariate associations, a mass difference network (MDiN) was created (Fig. 6c-e) in which nodes are the metabolite candidates and edges are formula differences (potential biochemical reactions). Fig. 6b visualizes the compound classes shown in the metabolite networks. As implied by the similarity of their loadings with the acute viral infection metabotype, the combined treatment with latent virus plus CNP showed high similarity to the pattern seen in acute virus infection, characterized by an upregulation of phospholipids, but not to the control groups (Fig. 6d and e and Additional file 1: Fig. S7).
More detailed information on the involved building blocks could be mined using mass difference enrichment analysis (MDEA; as described by Moritz et al. [42]). In MDEA, MDiN-edges are interpreted as biochemical reactions or building blocks, whose association to nodes of interest can be investigated by the discrete Fisher's exact test. MDEA results implied the induction of various fatty acid pathways as the major building blocks of acute viral infection and markers were C18-C22 saturated and poly-unsaturated fatty acids (Z-Scores and p-values ranged from 5.87 to 9.94 and 0 to 8.68 × 10−8, respectively). Confirming the data shown in the MDiN, one major upregulated compound class was found to be glycerophospholipids (see also Additional file 1: Fig. S7). Furthermore, arachidonic acid and eicosatrienoic acid molecular formula were determined significantly frequent, indicating a non-random usage of these substances and similar tendencies could also be found for other unsaturated fatty acids such as linoleic acid. On the other hand, down-regulated, or extensively consumed, metabolites were found to be composed of typical metabolites of glycolysis pathways. DWCNT exposure of latently infected mice caused a similar response to a certain extent, which was also accompanied by upregulation of phospholipids (Additional file 1: Fig. S8), but the response was not significant. This might be due to the strong hydrophobic nature of DWCNT which might adsorb compound groups such as phospholipids and therefore detract them from analysis. Taken together, the profound match of the metabolite pattern observed between acutely infected mice and latently infected mice treated with CNP suggests that – similar to our aforementioned observations – short-time treatment of latently infected mice with NP induces a boost of lytic virus replication and restores features of an acute virus infection in these mice.
Latently infected B cells and macrophages are differentially affected by TiO2 NP and diesel exhaust particles (DEP)
To investigate if other types of commonly investigated low-toxicity low-solubility particles show a similar effect as described for CNP and DWCNT, we exposed latently infected cell lines to aqueous dispersions of commercial titanium dioxide NP (TiO2 NP, P25) or to diesel exhaust particles (DEP, SRM 2975) (Z-Average and PdI see Additional file 1: Fig. S1). The cells were treated with TiO2 NP or DEP for 72 h and virus titers in the supernatant and the ratio of ORF50/ORF73 expression were determined. Differences between the cell lines concerning the impact of the different types of NP were detected. Significant effects on virus reactivation were, however, only observed for DEP and the expression of the lytic switch protein ORF50 in S11 B lymphocytes (Additional file 1: Fig. S9a and c). Under these conditions, TiO2 NPs induced a similar pattern but did not reach statistical significance (p > 0.05). For ANA-1 macrophages, neither NP caused significant virus reactivation, i.e. increased the production of lytic virus and the expression of the viral gene ORF50 (Additional file 1: Fig. S9b and d). These comparisons suggest that – depending on the cell type – different nanomaterials might have different potencies to affect the maintenance and control of viral latency.
Carbon NP reactivate EBV from latently infected human cells
Since both inhalation of environmental NP and persistent herpesvirus-infection have been implicated to contribute to the development of chronic lung disease, we hypothesized that the combination of both might lead to a different outcome than each factor alone. Given that virtually every human being is persistently infected with herpesviruses, NP exposure of an already infected individual can be easily envisaged as a practically relevant scenario. At present, no information about the health relevance of a suchlike liaison of NP and persistent virus infection is available.
There are several publications providing evidence that the presence of carbon-based NP has an influence on virus infection in mice or in cells. For example, it has been demonstrated that exposure of cells to single-walled carbon nanotubes increases the susceptibility of these cells to infection with influenza H1N1 [49]. Additionally, it has been shown that preexposure of mice to carbon black prior to infection with respiratory syncytial virus (RSV) induces an inflammatory milieu that promotes disease exacerbation [50]. Furthermore, treatment of RSV-infected mice with ultrafine carbon black enhances the expression of various chemokines that are associated with virus infection, and leads to an enhanced RSV-induced airway hyperresponsiveness to methacholine [51]. In this paper, we show that NP exposure of persistently herpesvirus-infected cells in vitro reactivates latent virus. Interestingly, we observed differences depending on the cell type (B cells, macrophages) and on the type of NPs (CNP, DWCNT, TiO2, or DEP). For example, the effect of DWCNT was more pronounced than the one by CNP in most of our experiments. This does not seem to be a consequence of the higher surface area but might be due to the fact that DWCNT, apart from inducing oxidative stress and activating acute inflammatory responses, affect a number of additional cytotoxicity pathways [52]. The triggers of herpesvirus reactivation and the underlying molecular mechanisms are – taken as a whole – only incompletely understood [53, 54], and along the same line, we so far can neither depict by which mechanisms/pathways NP might induce reactivation of latent virus nor which are the target cells for this interaction in vivo. In our in vivo experiments we could show that NP exposure of persistently infected mice leads to the expression of lytic viral proteins and restores a signature observed during acute virus infection. Although we observed the induction of lytic viral proteins after NP treatment of latently infected mice, no increase in infectious virus or virus genomes could be detected. This might indicate that only small amounts of new infectious virus were produced which were below the detection limits of the assays used. Another possibility is that NP treatment induces an abortive reactivation which leads to re-expression of lytic virus proteins – serving as potential targets for the immune system – but does not lead to the completion of the replication cycle and the production of new infectious virus. Such a repetitive appearance of viral proteins induced by NP-exposure might nevertheless provoke the infiltration of immune cells and finally cause a chronic aberrant inflammatory response even in the absence of completely assembled infectious virus. For example, it has been shown that the CD8+ T cell response against MHV-68 antigens can mediate inflammation and altered cellular recruitment to the lung, finally resulting in immunopathology and fibrosis [55]. The increased appearance of glycerophospholipids which was found by metabolome analysis in the lungs of acutely infected mice as well as in the lungs of latently infected mice treated with NP is another indicator for the presence of lytic virus replication – irrespective of successful completion of the replication cycle or not. Noteworthy, the phospholipid pattern was observed in association with the detection of lytic MHV-68 proteins only, but not with the acute inflammatory response caused by NP exposure alone. As shown by Sutter et al., who investigated the role of phospholipids in infection with Herpes simplex virus, virus infection triggers the production of phospholipids to maintain cellular membrane integrity and to deliver membrane components for envelopment of virus capsids and formation of transport vacuoles during virus production [56]. Other substances detected by metabolome analysis such as arachidonic acid, eicosatrienoic acid, and linoleic acid imply upregulation of mediator molecules of immunomodulatory and of oxidative stress related pathways after exposure of latently infected mice to CNP. Particularly arachidonic and eicosatrienoic acid have been described to have an impact on primary infection with herpesviruses as well as on virus reactivation [57].
In this paper, we show that exposure of latently infected cells or tissues to NP leads to reactivation of latent virus accompanied by an increase in viral proteins and metabolome- and transcriptome-signatures that can also be found in acute virus infection. Concerning the health relevance for humans it should be considered that application of a single dose of NP, as in our experiments, does only partially reflect the occupational settings where the applied amount might be gradually accumulated over one working week. Nevertheless, repetitive appearance of even a small amount of viral proteins, induced by exposure to NP, might be sufficient to trigger a chronic aberrant immune response and consequently lead to tissue damage. The question whether the combined exposure to NP and virus de facto causes disease aggravation needs to be further investigated and will be a focus of subsequent studies.
Nanoparticles used in this study
Size a [nm]
BET [m2/g]
Carbon black
Evonik Degussa GmbH
Carbon nanotubes
10 × 1000
Evonik Degussa GmbH
Diesel exhaust particles
Cell lines
BHK-21 cells (ATCC: CCL-10) were grown in Glasgow-MEM (PAN Biotech, Aidenbach, Germany) supplemented with 5% fetal calf serum (FCS; PAN Biotech, Aidenbach, Germany), 5% tryptose phosphate broth, 2 mM L-glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin. NIH 3T3 cells (ATCC: CRL-1658) were grown in DMEM High Glucose (Gibco, Darmstadt, Germany) supplemented with 5% FCS, 2 mM L-Glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin. The persistently with MHV-68 infected B cell line S11 [30], the two macrophage cell lines ANA-1 [60, 61] and MH-S (ATCC: CRL-2019), and human LCL cell lines (kindly provided by Bettina Kempkes and Josef Mautner, Helmholtz Zentrum Muenchen, Munich, Germany) were cultivated in RPMI (Gibco, Darmstadt, Germany) supplemented with 15% fetal calf serum (FCS; PAN Biotech, Aidenbach, Germany), 2 mM L-glutamine, 1% non-essential amino acids (Gibco, Darmstadt, Germany), 100 U/ml Penicillin and 100 μg/ml Streptomycin. For S11 and MH-S cells, 50 μM 2-Mercaptoethanol (Bioconcept, Allschwil, Switzerland) was added to the medium.
Determination of cell viability by WST assay
Cell viability after exposure to NP was measured by WST assay, which determines the activity of mitochondrial succinate dehydrogenase in cells, according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Briefly, cells were plated and incubated with the indicated concentrations of NP for 72 h. WST reagent was added and incubated with the cells for 2 h at 37 °C. The plates were centrifuged to remove the bulk of NP agglomerates and supernatants were transferred to a new plate prior to analysis. Enzymatic conversion of WST reagent was determined using an ELISA-reader at 430 nm with 630 nm as reference.
Analysis of lytic virus growth in vitro after NP treatment
To test lytic growth of MHV-68 in vitro after NP treatment, MH-S cells or LA-4 cells were infected with a MOI of 1 for 2 h. After removing the inoculum (=0 h), cells were washed two times with PBS and then treated for 2 h with 50 μg/ml NP (NP preparation see section "Nanoparticles"). After the incubation period, the inoculum was removed, the cells were washed two times with PBS and then incubated with fresh medium at 37 °C and 5% CO2 until the supernatants or the supernatants together with the cells were harvested at different time points after infection. Virus titers were determined by plaque assay on BHK-21 cells.
In vitro assay for measurement of low level virus replication in infected macrophages
To measure low-level virus replication in infected macrophages, a modification of a previously described method for a limiting dilution in vitro reactivation assay was used [28]. Briefly, MH-S cells (alveolar macrophage cell line) were plated in a 6-well plate and infected with MHV-68 o.N. at an MOI of 0.01. The inoculum was removed and each well was washed two times in PBS. The cells were then either left untreated or incubated with 50 μg/ml CNP or DWCNT (NP preparation see section "Nanoparticles"). After 2 h, the medium containing the NP was removed and each well was washed two times with PBS. The cells were incubated for 1 min with an acidic citrate buffer (pH = 3.0) to remove remaining lytic virus from the inoculum and washed three times with medium. Serial threefold dilutions of infected MH-S cells were plated on monolayers of 7 × 103 low-passage NIH 3T3 cells per well in 96-well tissue culture plates. Twenty-four wells were plated per dilution (starting with 1 × 103 MH-S cells). NIH 3T3 cells were screened microscopically for a viral cytopathic effect for up to 2 weeks. To differentiate between freshly produced virus and residual lytic virus from the inoculum, serial threefold dilutions of MH-S cells were plated before or after mechanical disruption of viable cells (by two freeze-thaw cycles). Frequencies of cells supporting lytic virus replication were calculated on the basis of the Poisson distribution by determining the cell number at which 63.2% of the wells scored positive for CPE. To compensate for variations in the infection efficiency, the viral genomic load was determined as described below and taken account of when calculating the frequency of cells producing lytic virus.
Generation of a persistently infected macrophage cell line
To generate a persistently infected macrophage cell line, we constructed a recombinant MHV-68 containing a hygromycin-resistance cassette by a two-step mutagenesis procedure using the BAC-technology [62, 63]. To this end, a 2.4 kb expression cassette containing the coding sequence of hygromycin phosphotransferase driven by the SV40 early-enhancer promoter, was excised from vector pRTS-1 [64] (kindly provided by Bettina Kempkes, Helmholtz Zentrum Muenchen, Munich, Germany) and cloned blunt end into the PmlI site (nucleotide position 46.347 of the MHV-68 genome) of the plasmid pST76K-SR already containing a 4.0 kb SphI-SacI fragment of MHV-68 (nucleotide positions 44.301 to 48.346). As a result, the hygromycin phosphotransferase expression cassette is flanked on both sides by homologous sequences as needed for homologous recombination during the two-step mutagenesis procedure. BHK-21 cells were transfected with 1.5 μg of BAC MHV-68-Hygro DNA using X-treme GENE HP DNA Transfection Reagent (Roche, Mannheim, Germany) to reconstitute recombinant MHV-68-Hygro. A virus stock was generated and the virus titer was determined by plaque assay on BHK-21 cells. To establish a permanently infected cell line, the bone marrow derived macrophage cell line ANA-1 was infected with MHV-68-Hygro at an MOI of 1. Hygromycin B (Sigma-Aldrich, Seelze, Germany) at a final concentration of 500 μg/ml was added 24 h after infection and persistently infected cells were expanded under permanent selection. As the BAC-sequence in the virus genome contains the GFP-gene, the cells could be monitored under the fluorescence microscope and more than 90% of the cells proved to be GFP positive.
Treatment of persistently infected cell lines with NP
To analyze the effect of NP exposure on persistently infected murine cells, the B cell line S11 and the macrophage cell line ANA-1/MHV-68 were used. To investigate the effect of NP on persistently infected human cells, human lymphoblastoid cell lines (LCL) were used. NP were suspended as described above and added to the cells at a concentration of 5 μg/ml or 50 μg/ml. After 72 h, supernatants were harvested for analysis of virus titer by Plaque assay (murine cells) or qPCR (human cells), and cells were harvested for RNA isolation and subsequent RT-PCR.
Measurement of viral genomic load by quantitative real time PCR
DNA was isolated from lung tissue samples that were homogenized by using the FASTPREP®-24 instrument (MP Biomedicals, Heidelberg, Germany), from cell culture supernatants, or from infected cell lines with the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany). The viral genomic load in infected murine cells or in murine lung tissue was determined by quantitative real-time PCR using the ABI 7300 Real Time PCR System (Applied Biosystems, Foster City, CA) as described previously [65]. The amount of viral genomes in cell culture supernatants from LCL was analyzed by real time quantitative PCR for the viral gene EBNA1 using the Taqman SYBR green PCR master mix (Applied Biosystems, Foster City, CA).
Primer sets used in this study
Gene name
Forward primer (5'–3')
Reverse primer (5'–3')
In vivo experiments
C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany) and housed in individually ventilated cages (IVC) during the MHV-68 infection period. Mice were anesthetized with ketamine and xylazine and infected i.n. with 5 × 104 PFU MHV-68. After 28 days, mice were either left untreated or instilled with 50 μg of spherical (CNP) or double-walled fibre-shaped carbon nanoparticles (DWCNT) per mouse as described earlier [5, 58] (NP preparation see section "Nanoparticles"). Lung tissue was harvested after 24 h for transcriptome and metabolome analysis as well as for determination of viral genomic load and for histology. Bronchoalveolar lavage (BAL) cells for RNA isolation and analysis of viral gene expression were collected by cannulating the trachea and rinsing the lung six times with PBS as described earlier [26]. All animal experiments were in compliance with protocols approved by the local Animal Care and Use Committee (District Government of Upper Bavaria; permit numbers 124/08 and 67/2015).
For the metabolomics analysis, lung tissue samples (three mice per group) were processed by using the FASTPREP®-24 instrument (MP Biomedicals, Heidelberg, Germany). 50 mg of lung tissue were homogenized in 1 ml of ice-cold methanol (LC/MS grade, Sigma-Aldrich, Steinheim, Germany). The homogenates were centrifuged at maximum speed in an Eppendorf centrifuge to remove debris. The supernatants were stored at −80 °C and diluted 1:50 in methanol prior to metabolome analysis. Samples were injected in a randomized order at a flowrate of 120μLh−1 using a Gilson autosampler system (Gilson, Inc., Meddleton, WI, USA). Electrospray ionization (ESI) was performed using an APOLLO II ion source (Bruker Daltonics GmbH, Bremen, Germany) in negative ionization mode with capillary voltage and spray shield voltage being set to −3000 V and 500 V, respectively. Drying gas flow rate and temperature were set to 4Lmin−1 and 200 °C. The nebulizer pressure was set to 1.1 bar. Ultra-high resolution and accuracy mass spectrometra were recorded using a Bruker (Bruker Daltonics GmbH, Bremen, Germany) solariX Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR-MS) equipped with a 12 T superconducting magnet. External calibration of the mass spectrometer is performed daily using a 1 mgL−1 arginine/methanol solution. Linear calibration on at least 4 arginine clusters is accepted once the standard deviation of m/z error was minor 100 ppb. Mass spectra were recorded over a mass range of 129 m/z to 1000 m/z. The time domain was set to 4 M words (MW) and the resolution at m/z = 400 was R = 430,000.
Raw mass spectra were pre-processed using Data Analysis Version 4.1 (Bruker Daltonics GmbH, Bremen, Germany). All mass spectra were uploaded and peak picking was performed using a signal to noise cutoff of S/N = 4. Linear calibration was performed using an in-house generated list of 390 metabolite m/z values that are frequently occurring across species and bio-fluids. The central error-m/z distribution was visualized within the Data Analysis calibration functionality and centered on zero ppm given a linear calibration function. The overall error standard deviation was found to be < 100 ppb. All calibrated mass spectra were exported as ASCI file and aligned with the in-house written software 'Matrix Generator' given an error window of 1 ppm.
M/z signals which were not once detected within a full triplicate were omitted. M/z peaks were subjected to combinatorial formula assignment at ±0.5 and given elemental counts of C1-100O0-70N0-20S0-3P0–3 using an in-house written software. Masses were filtered for the Senior rules and for diverse approximations of elemental relationships. Formulae were then run through a strict isotopic pattern matching algorithm assuming infinite resolution and given a noise level that was set to be the minimum of all maximal m/z intensities across all samples. Validated formulae were used as starting points for mass difference network-based formula assignment to low abundance peaks (> > 95% of a dataset) [66]. The initial dataset of >200.000 m/z values was reduced to 6890 m/z values by removal of non-triplicate features and reduced to 2697 sum formula annotations.
Data normalization was performed as follows: The inter quartile ranges (IQRs) of all non-zero entries per sample were calculated. The sample-wise Euclideans of all m/z feature intensities that were elements of their corresponding IQR were calculated and used for normalization.
Data was separated into two data sets which were composed of the second hit experiments (Virus 28d + CNP 24 h or Virus 28d + DWCNT 24 h), their corresponding time matched controls and a mouse-group with acute virus infection (Virus 6d).
Both datasets were scaled by Z-transformation and subjected to principal component analysis (PCA) using the Perseus software, version 18.104.22.168. The scores of the first three components of each dataset were tested for significant differences between combined exposure, single exposures and untreated controls using Student's T test in Microsoft Excel 2016. Mass difference networks were reconstructed and mass difference enrichment analyses were performed using Matlab R2011 (as described in detail by Moritz et al.[42]). The theoretical mass difference network was reconstructed using 490 reaction equivalent mass differences (REMDs), part of which were derived from the KEGG metabolic maps and part of which were a manually curated extension and correction of data published previously by Breitling et al. [67]. Given theoretical molecular masses derived from molecular formula assignment, REMDs, which here are interpreted as building blocks, were used as edges to connect all annotations (nodes). REMDs were then tested for significant associations to metabolic features (molecular formulas) of interest using Fisher's exact test, which assumes a hypergeometric distribution. This technique is used to include features that could not be matched to e.g. the human metabolome database (HMDB [41]). Box-plots were generated using ggplot2 in RStudio Version 0.99.489. Networks were visualized using Gephi-0.8.2.
Immunohistochemical staining for lytic virus proteins
Expression of MHV-68 lytic proteins in the lung of NP-treated or control animals was examined by standard immunohistochemical methods. Since all lungs were divided for different assays, collapsed lungs had to be used for histology. Lung tissue was embedded in paraffin and cut into 4 μm sections. Slides were incubated with 3% hydrogen peroxide to bleach tissues and produce a better contrast for the alkaline phosphatase staining procedure. Following epitope retrieval by heating the sections in citrate buffer (pH = 6.0), the sections were incubated with blocking buffer (Rodent Block M; Biocare Medical/Zytomed Systems, Berlin, Germany) and labeled with a polyclonal rabbit serum directed against lytic proteins of MHV-68 (described previously by Steer et al. [68]; 1:500 dilution). After washing, Rabbit-on-rodent-AP-polymer was added (Biocare Medical/Zytomed Systems, Berlin, Germany), and finally, the phosphatase reaction was visualized using the Vulcan Fast Red Chromogen Kit (fuchsin-red reaction product; Biocare Medical/Zytomed Systems, Berlin, Germany). All sections were counterstained with hematoxylin.
Transcriptome analysis
Total RNA from lung tissue was isolated employing the RNeasy MiniKit (Qiagen, Hilden, Germany) including DNase digestion of remaining genomic DNA. The Agilent 2100 Bioanalyzer was used to assess RNA quality and only high quality RNA was used for microarray analysis. 300 ng of high quality total RNA were amplified using the Illumina TotalPrep RNA Amplification kit (Ambion, Life Technologies, Carlsbad, CA, USA). Amplified cRNA was hybridized to Mouse Ref-8 v2.0 Expression BeadChips (Illumina, San Diego, CA, USA). Staining and scanning were done according to the Illumina expression protocol. Data was processed using the GenomeStudioV2010.1 software (gene expression module version 1.6.0) in combination with the MouseRef-8_V2_0_R3_11278551_A.bgx annotation file. The background subtraction option was used and an offset to remove remaining negative expression values was introduced. Data normalization (quantile) was performed by utilizing the statistical programming environment R implemented in CARMAweb [69, 70]. Genewise testing for differential expression was done employing the limma t-test and Benjamini-Hochberg multiple testing correction (FDR < 10%). Heatmaps showing genes that were at least 1.5fold regulated in mice treated with latent virus and NP compared to untreated control mice were generated with CARMAweb. Pathway enrichment analyses were done with the Ingenuity pathway analysis software (IPA®, Qiagen, Redwood City, CA, USA, https://www.qiagen.com/ingenuity). For genes that were detected by more than one probe, only one representative value is shown. Array data has been submitted to the GEO database at NCBI (GSE79501).
Statistical methods
Datasets were analyzed by Student's t-test using the GraphPad Prism software, vs5 (GraphPad Software, Inc., San Diego, CA, USA). Results with a p-value < 0.05 were considered significant. Statistical analysis of transcriptome data was performed as described in the section "transcriptome analysis".
Bronchoalveolar lavage
Carbonaceous nanoparticles
Cytopathic effect
- d:
Diesel exhaust particles
Double-walled carbon nanotubes
- h:
Human cytomegalovirus
Idiopathic pulmonary fibrosis
Kaposi's sarcoma-associated herpesvirus
Lymphoblastoid cell line
Mass difference enrichment analysis
- MDiN:
Mass difference network
Murine gammaherpesvirus 68
Principal component analysis
Reverse transcription polymerase chain reaction
Tissue culture infective dose (amount of a pathogenic agent that will produce pathological change in 50% of cell cultures inoculated)
- TiO2:
Titanium dioxide
We are grateful to the members of our animal facilities for expert technical assistance. We also thank the members of our institutes for helpful discussions.
This work was supported by intramural funding for Environmental Health projects of Helmholtz Zentrum München – German Research Center for Environmental Health to T.S., H.A. and P.S.-K., and by grants from the Helmholtz Portfolio Theme 'Metabolic Dysfunction and Common Disease' and the Helmholtz Alliance 'Imaging and Curing Environmental Metabolic Diseases, ICEMED' to J.B.
Availability of data and materials
Additional supporting data are shared as supplementary material: Additional file 1 (.pdf), Additional file 2 (.xlsx).
Microarray data has been submitted to the GEO database at NCBI (GSE79501).
Authors' contributions
C.S. designed and performed the experiments, analyzed the data, wrote and edited the paper. F.M. designed and performed the metabolome analysis, analyzed the data, wrote and edited the paper. S.C., B.S. and D.S. performed the experiments. M.I. and J.B. performed microarray analysis and analyzed the data. O.E. participated in coordination of the study and acquiring funding. P.S.-K. participated in acquiring funding, supervised metabolome analysis and gave conceptual advice. H.A. and T.S. participated in acquiring funding, supervised the project, designed the experiments and analyzed the data, wrote and edited the paper.
Competing interests
The authors declare no competing financial interest.
Consent for publication
Not applicable.
Ethics approval and consent to participate
All animal experiments were in compliance with protocols approved by the local Animal Care and Use Committee (District Government of Upper Bavaria; permit numbers 124/08 and 67/2015).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors' Affiliations
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text:
Discussion in 'Colorado Football Message Board' started by The Guest, Jan 3, 2012.
Does that mean that Nebraska was 2 plays from winning?
Not by my math.
On the other hand, I believe that Bo makes a compelling point. Let the record books show that nebraska was the better team, regardless of the outcome.
how very HaLkish.
Our old buddy Mark Knudson wrote a column about the fuskers being a better team that SC, and pretty much blames the refs for the loss. Not to mention excusing Alonzo Dennard's Mike Tyson impersonation by blaming it on the classlessness of SC.
Disagree. While HaLk was quick to point out that the Buffs were a few plays away, he never went so far to say that we were the better team.
Solid point. However, the douche and stupidity levels are high enough to warrant the insult.
Wow! I'm not even annoyed with that guy, I'm flat-out embarrassed for him. Just, wow!
some pretty classic comments in the comment section. Even fusker fans are telling Knudson to shut his yap.
That's just embarassing....I am so glad we don't share a conference with crybabies like that anymore. CU got their fannies kicked week in and week out this year, and I don't recall anybody here crying and making excuses like that. Pathetic.
I think 'tini did.
I'm sure DBT bitched about the refs.
I watched the game and once SC caught that hail mary for a TD before the half I never thought neb was going to win
The refs tried to give the game to Utah.
I thought we should have won that game.
Knutson is still writing? I didn't recognize the paper. Where is that?
Not to mention Oregon. They called us for 10 penalties. I'm pretty sure we were the better team that day, they just didn't give us a chance. Not to mention their classless, big mouth players. You couldn't blame our guys for getting some calls against us...
It's probably not exactly a paper, and no doubt the only "media outlet" that would still allow Knudson to pollute the interwebz with his drivel...
Only when they deserve it! But I haven't blamed them for a loss since the bogus call when we played Miaim at Folsom.
Not even in Ames in 2007?
by the way here is news flash, Bo P suks and the way he throws his temper tantrums, he fits in at nu
That's the one I was thinking of. Go screw, Clete Blakeman.
Oh yeah. Forgot about that. And the ref was a fusker no less. Oh the irony.
I'm surprised he didn't mention the fuskers MNC's in the article, what a loser.
And, being Pac-12 quality, they failed.
I flashed back to that game when Wisconsin made their big first down catch. I knew they weren't going to have enough time to run that play if we didn't. That Wisc coach should have known to call a real play. I do (now)
Schooled by a high school student. :lol:
I'd really like to see a Meltdown Nebraska Style thread.
Paging BamaCharlie....
Blame it on the refs - the oldest and most common excuse for a loser. Nebraska will never ever be what is was back in the 90's - those days are gone with Osborne.
That's an excellent response.
Separate names with a comma.

subset: commoncrawl


text:
Pirates on Friday hijacked a Spanish fishing vessel in the Indian Ocean about 600 km off the Somali coast, Basque regional agriculture and fishing official Pilar Unzalu said.
MS Alakrana, which is based in the Basque port of Bermeo, informed another fishing boat of its situation, following which the European Union's Operation Atalanta launched a security operation.
Two Luxembourg planes flying over the vessel observed armed people on board.
The company which owns the Alakrana said it had been unable to contact the 36-member crew, which comprises 16 Spaniards, eight Indonesians, four Ghanians, three Senegalese as well as two people from Ivory Coast, two from Madagascar and one from the Seychelles.
Sources of the Basque tuna fish sector said the Alakrana was hijacked in international waters and was being taken towards the Horn of Africa country.
On September 4, the Alakrana managed to escape from would-be hijackers.
Josu Erkoreka from the Basque Nationalist Party (PNV) criticised Defence Minister Carme Chacon for not placing Spanish marines on board Spanish vessels fishing in the Indian Ocean.
Prime Minister Jose Luis Rodriguez Zapatero's Socialist Party rejected the accusations, saying such a measure was not judicially possible.
Nearly 20 Spanish fishing vessels were in the same area as the Alakrana at the time of the hijacking.


text:
Hacking Game
This project was awarded to xenonartws for $5014.99 USD.Get free quotes for a project like this
Project Budget$500 - $5000 USD
Total Bids2
Project Description
This is a (relatively) simple browser-based game. The players compete to "hack" websites around the web. The objective of the player is to hack as many web sites as he can, these websites being as famous as possible.
The player view is a frameset consisting of a top frame (an HTML "toolbar" like [url removed, login to view], not a browser plugin) and a lower frame, which is any website the player wants to hack.
See the attached detailed specifications for some sample mockups and detailled explanations.
## Deliverables
* A set of unit tests covering 100% of the code written or modified for the job. There is no need to cover the code that already existed and that was not modified.
* A single command without argument must run all the unit tests, report the total percentage of coverage and list all the lines of code that are not covered.
* Implement a verbose mode for running the tests so identifying which unit test fails and why is straightforward.
* All javascript functions, if any, must be tested using [url removed, login to view] and reach 100% coverage. All code must be encapsulated in functions. All javascript functions must be defined in .js files.
* The tests must be implemented using the same techniques as the pre-existing tests of the software being modified. It is not acceptable to create a new test environment.
Awarded to:
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Susan Howick
Personal Statement
Susan Howick is a Professor of Management Science. She gained a BSc in Mathematics from Dundee University and a MSc in Operational Research and a PhD in Management Science from Strathclyde. Her main research interest lies in the use of System Dynamics simulation. A particular focus of her work has been using System Dynamics to explore the behaviour of large complex projects and developing systemic risk analysis tools. Susan's work often involves the integration of multiple modelling methods where the aim is to provide robust, client-orientated decision support tools.
Susan was President of the Policy Council for the UK Chapter of the System Dynamics Society in 2007-08. She won the Operational Research Society Goodeve medal for her joint paper with Colin Eden "The Impact of Disruption and Delay when Compressing Large Projects: Going for Incentives?" 2001. Susan is a member of the Publications Committee of the System Dynamics Society. Her consultancy experience includes projects for Bombardier, Strathclyde Police, Reed Elsevier, PriceWaterhouse Coopers, Scottish and Southern Energy.
Research Interests
My research interests include how multiple modelling methods can be integrated effectively to create robust, client-orientated decision support tools. I have been involved in creating such tools for clients in healthcare, engineering, construction and the energy sector. In addition, I have an interest in understanding what causes complex projects to overrun and in developing new risk assessment and management tools, which enable risk systemicity to be considered.
Teaching Interests
Susan has 20 years of experience in teaching to undergraduate, postgraduate and post experience students. Programmes on which she regularly teaches are MBA, MSc Business Analysis & Consulting and MSc Operational Research. Her teaching includes general Management Science classes in additon to specialised classes in system dynamics and project risk management that reflect her key research interests. Susan has delivered training courses for organisations such as Bombardier Inc., Babcock, Scottish and Southern Energy and Scottish Government. She has also taught extensively via distance learning and was involved in developing two unique online MSc's.
Expertise & Capabilities
Susan's research interests are in system dynamics modelling, the integration of modelling methods, disruption and delay analysis on complex projects, applying systems methods to project risk management
A toolkit of designs for mixing Discrete Event Simulation and System Dynamics
Research output: Contribution to journal › Article
Stalking resilience: cities as vertebrae in society's resilience backbone
Research output: Chapter in Book/Report/Conference proceeding › Conference contribution
Learning from mixed OR method practice: the NINES case study
Research output: Contribution to journal › Article
34th International System Dynamics conference
Activity: Participating in or organising an event › Key-note speaker and plenary lectures at conferences
External Examiner for PhD thesis for student at Indian Institute of Technology Kharagpur
Activity: Other › Types of External academic engagement - External Examiner
Invited speaker at seminar at Manchester Metropolitan University
Activity: Other › Types of External academic engagement - Invited talk
H2020 - Smart Mature Resilience
Project: Research
Doctoral Training Grant 2006 | Morgan, Jennifer
Project: Research Studentship - Internally Allocated › Research Studentship (Internally Allocated)
STRATHCLYDE - ESRC STANDARD RESEARCH TRANSITION STANDARD QUOTA DTG | Scotland, Alistair
Project: Research Studentship - Internally Allocated › Research Studentship (Internally Allocated)
Elected member of the Policy Council of the International System Dynamics Society
Prize: Election to learned society
Elected member of Policy Council of the UK Chapter of the System Dynamics Society: Elected
Prize: Election to learned society
Elected to Publications Committee for International System Dynamics Society
Prize: Election to learned society


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These are the Highest Quality Art History Tutors in Fish Hoek, Cape Town. Get Art History Lessons in your home with Teach Me 2 – the Best in Tutoring
I have a BA in Theory of Art (Rhodes University, 1993-1996).
I did Art History until Grade 12 and Art Philosophy in First Year at University.
I have a good grasp of the critical thinking required to assess all forms of Art.
I have 2 years experience teaching Visual Art to Grades 10 and 11.
I have a great love for art and art history, and obtained a distinction for both Art and Graphic art in Grade 12. It would be great to share my love and knowledge of the subject.
I took visual arts from grade 10 to 12, maintaining marks above 75%, ending with an A in Matric. specific help for Art History essay writing.
As mentioned above, I am very passionate about Art and one of my favourite sections within the subject Fine Art is Art History. I can tutor at grade 12 level as I have done so before and I know what the syllabus entails.
I was involved in Visual Art tutoring at school.
It is a subject that I am very confident in and have further developed the necessary skills for during an Art History course as well as studying Film and Media at university. I have experience tutoring it at a Matric level.
I received a Distinction for Art in Matric, particularly excelling in art History, I often receiving 100% for essays.


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Played 8 courses up the East coast of Scotland and this was my favorite. Very playable, great views and friendly staff
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Played 8 courses up the East coast of Scotland and this was my favorite. Very playable, great views and friendly staff
Played 8 courses up the East coast of Scotland and this was my favorite. Very playable, great views and friendly staff
The problem with lots of the best courses is they think your honoured to be there - and while that's true to a certain extent, you want to feel like they want you there - Castle Stuart does just that - I've played about 40 of the top 100 courses and the welcome given today was second to none -... More
It's expensive, but it's sure worth it. One of the finest links courses I've ever played. Home of the Scottish Open recently. A number of holes play right along beautiful Moray Firth (w/ a look back @ Inverness). Great clubhouse & staff. Superb practice putting greens. Pray for good weather, as either the rain and/or wind will play havoc on... More
Booked three course Highland hat trick deal Played Castle Stuart as our first course. We were the last out on the course and had it to ourselves. Hired electric buggies - £20 each which was a little expensive - £8 more than at the other courses we played. The club house is very nice with great views over the Moray... More
The course is spectacular and the views breathtaking, but that won't surprise many familiar with this course. Where this place really excels is in the extra touches. Bare in mind you've likely paid three figures for your round here but unlike Gleneagles they don't fleece you everywhere else. From unlimited range balls, free trolleys and a free water bottle vending... More
Fine club house and friendly reception staff. Course has several tees so length can be varied, helpful in the generally strong winds. Course in good condition and with mostly fine views. Generally challenging and better suited to the player with a reasonable level of skill and experience. Expensive by local standards and let down as an overall experience by very... More
I had heard very good reports from friends and family playing this relatively new links course to the east of Inverness so the opportunity to play could not be missed. The layout is sensational with holes running alongside the Moray Firth in both the first and second nine. Holes carved out of the dunes some 100 to 200 feet above... More
What a fantastic course that should be played by everyone who loves to golf. As soon as you arrive, the staff are there to greet you, grab your clubs, introduce you to your caddies and give you your welcome gift. A very high quality establishment. The course was in great shape and the views are spectacular. The Caddie Master suggested... More
Least memorable of all the courses played in 2 week golf tour. Do not bother going out of your way to play here. If you happen to be in the Inverness area, keep going better and less expensive options nearby.
From the moment you arrive you know you are in an establishment of superb quality. This isn't just playing a round of golf. The staff are fantastic and guide through the clubhouse, ranges, course start. Everything is pristine. The course itself is terrific and a fitting venue for the Scottish Open. Finally the food and bar in the clubhouse are... More
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Photo by: Leafpoo…
Leafpool01's Photos
True Bug
I don't know exactly what kind of insect this is but I know it is some kind of true bug:)
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Category: Bugs / Insects / Spiders
Comments for this photo
March 25, 2017
What is a true bug?
March 25, 2017
@sarsha, it's a type of insect identified by its wings, which cross into the shape of an "x".
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Online Banking is currently down for maintenance. You can click here to view your deposit account balances and transactions. We expect to be back online shortly. Thanks for your patience.
SunTrust Online Cash Manager is undergoing scheduled maintenance and is temporarily unavailable. We apologize for the inconvenience. Please try again later.
As you near retirement take the time to think about your most important priorities. This exercise can help you target your spending and saving in accordance with what you want to achieve—now and in the future.
Staying on track is more important than ever when you near the end of your working career and have less time to set aside savings.
"Setting specific goals provides a road map that shows you what you need to do to keep your financial plan on track," says John Lopez, who teaches personal finance at the University of Houston's Bauer College of Business.
But how do you start? The following steps can help:
Identify specific goals
Your goals have two components: what you want to achieve, and the amount of money you need to achieve them.
"Be specific," Lopez says. "Saying you want to retire at 60 is too broad. It's more effective to specify the amount of money you need in the bank to reach that goal."
That specificity makes it easier to determine what steps you need to take to reach your financial goals—and for how long. Say you're behind in your retirement savings. With 10 years until you hope to retire, you want to add $100,000 to your retirement savings. That means you have to set aside $1,000 a month for 10 years.
Set your priorities
When you have defined your financial goals, including how much money you need to reach each one and the time in which you'd like to achieve them, you can determine which goals get top priority.
"Your goals are competing for limited resources—your money—so you have to think about which to save for first," Lopez says.
At the top of your list should be basic financial preparedness, including establishing an emergency fund, paying down high-interest debt and saving for retirement or healthcare-related expenses. You also might want to make a plan to handle post-retirement healthcare expenses. For instance, you may want to draw up an estimated budget for retirement healthcare costs, or consider purchasing long-term care insurance coverage before you retire. Contributing to a child's or grandchild's college savings fund can go farther down the list.
Make it automatic
"Pay yourself first" by automating your savings. For example, if you're eligible, you're probably already contributing to your workplace retirement plan, which automatically deducts your retirement savings from your paycheck. (It's a good idea to contribute at least enough to qualify for any employer matching funds.)
And people who are 50 and older can add to their retirement savings with catch-up contributions. These allow you to contribute an additional $5,500 to 401(k) plans, and $1,000 to traditional and Roth IRAs.
Schedule an annual review
Evaluate your goals each year to adjust for any changes such as a remarriage, or a decision to stay in the workforce longer. It's smart to review your portfolio's asset allocation its mixture of stocks, bonds and cash investments with your SunTrust advisor at least once a year. Doing so can help determine whether you need to re-allocate your portfolio to better fit your retirement age and financial goals.
This content is educational in nature and is not an advertisement for a loan or business solicitation. It does not constitute legal, tax, accounting, financial or investment advice. You are encouraged to consult with competent legal, tax, accounting, financial or investment professionals based on your specific circumstances. We do not make any warranties as to accuracy or completeness of this information, do not endorse any third-party companies, products, or services described here, and take no liability for your use of this information.
Whether single by choice, through loss of a spouse/partner or as a result of divorce, 9 out of 10 women will at some point in their life be solely responsible for their financial wellbeing.
Answer key questions related to achieving your retirement goals.
For many Americans quickly approaching retirement, the question of whether or not to continue to work is a big one—possibly the biggest.
Erin Lowry, personal finance blogger at BrokeMillennial.com, shares her tips on how to approach retirement savings in your 20s.


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Title: (It Was Okay) Until You Walked Away. (1/1)
Rating (overall): PG-13
Pairings: Kurt/Blaine.
Spoilers: 03x05
Warnings: Bad language. Alcohol.
Word Count: 2,415
Notes: Inspired by the recently shared spoilers for Episode 5.
Summary: It was all okay, or at least it could have been, until Blaine walked away.
"Well, excuse me for trying to be fun and spontaneous!" Blaine bites out harshly, his jaw fixed firmly as his selfish words slip out of him in one long, spiteful slur. Clambering from the car as the words tumble out of him, smacking his head on the way, his judgment clouded by far too much alcohol, a blaze of bitter humiliation and a pool of burning embarrassment. Blaine's upset, so it doesn't come as a surprise to either of them when he coldly shrugs away from Kurt's reaching fingertips. It's even less of a surprise when he inches away from his boyfriend again and again and again.
Kurt's desperately trying to touch him now, he's trying to say something, something important, something like: 'I just wanted it to be perfect, Blaine. It's not that I don't want you, it's that I don't want you like this.' But Blaine is either too drunk or too upset to notice the subtle shift in Kurt's face and the frantic pleading in his eyes.
There is one thing they can agree on, though, even in the heat of the moment, even with their hearts pounding in their chests, and that's the fact that they both know that tonight was never supposed to end like this, tonight was supposed to be about them having fun as a couple. It was supposed to be about them dancing together, in public, without fear of being attacked or abused or hurt. Tonight was supposed to be completely liberating. It was supposed to be something beautiful. Unforgettable, even. It was never meant to be feel so ridiculously heavy; it was never meant to suffocate them both within an inch of their lives.
It's not long before a distraught Kurt has run out of ways to say, 'Please, Blaine, just stop this.' and a confused Blaine has stormed away, his aching feet falling haphazardly against the concrete, as he stumbles off into the distance. His body burning from the hours of dancing and laughing and kissing that proceeded this fallout. Blaine's almost completely out of sight by the time he realises that he can still hear Kurt calling out to him, his feet are heavy and his head is fuzzy as his boyfriend's voice cuts straight through him, "Blaine! Blaine Anderson, you stop right there! Blaine, please! Don't you leave me here!"
But Blaine is far too stubborn to stop, even though his heart twinges at the thought of Kurt standing by his car all night- his fingertips reaching out into emptiness. He's just feeling so embarrassed right now and that alone makes everything else seem so much worse. It gives everything a new, tainted edge. And all he can think about is the fact that he made a very serious pass at his boyfriend tonight, he'd offered Kurt the most intimate, personal part of himself and Kurt had thrown it straight back in his face. He'd asked Blaine to stop in a horribly low voice and Blaine's pretty sure that he's never been so mortified, he's never felt so stupid and that's really saying something. He's performed in theme parks. He's just never felt as utterly ridiculous as he did in that heart-stopping moment- Kurt's zipper halfway down, his hand halfway in his pants, as he whispered, desperately against Blaine's lips, 'No. Blaine. Not like this. Not when you wont remember a thing in the morning."
Blaine flinches at the memory and he's barely turned the corner, his angry footsteps wavering, when his pocket starts vibrating. The soft sound of 'Teenage Dream' mocking him horrifically with it's notions of 'going all the way tonight' and he knows exactly who it is, he knows it's Kurt because that's Kurt's special ring tone but he doesn't answer it, he can't deal with it, not right now, so, instead he pulls his phone out and slams an angry finger down to reject the call. He's been rejected tonight. It's only fair that Kurt is rejected too. It's only fair, he tells himself forcefully... but even he's a little dubious about that because it's not Kurt's fault, is it? It's not Kurt's fault that he doesn't want to lose his virginity, via a drunken fumble with his boyfriend, in the backseat of a car- a car that's parked behind a fucking gay bar. Even Blaine, in his drunken state, can see how that goes against every romantic bone in Kurt flawless body. In fact, when he really thinks about it it's fucking disgusting. It's not disgusting because of the location.. it's disgusting because it's just not them.
Kurt and Blaine don't usually do things like fool around in the back of cars or go to gay bars. Kurt and Blaine make each other romantic dinners and give each other soft, warming massages- bathed in soft candle light. They sing songs together and bake trays of cookies as they laugh about their friends and their days. They enjoy each other's comfortable and familiar company and they take the time to enjoy each other. They don't do things like proposition each other in car parks. Sure, that might work for some people and that might tick all the boxes for couples who live for spontaneity but that's just not them. It's nowhere near compatible with who they are as people or as a couple.
Blaine bites down on his bottom lip. Then his feet stop moving and he's left standing awkwardly on the concrete, his brain slowly kicking back into gear. As a conversation he had with Kurt a couple of months ago starts rushing through his head with the devastation of an unexpected hurricane:
"Blaine?" Kurt had started nervously, pushing his homework aside for a moment as a soft blush bloomed on his cheeks, "When we have sex... do you think... do you think it'll be beautiful?" It was no longer a case of if they had sex but when. Over the summer they'd carefully and lovingly overcome many of Kurt's intimacy issues and they both knew that it was only a matter of time. They both wanted it now. They both wanted somethng more. It just had to feel like the right time.
Blaine had stopped reading when the unexpected question hit his ears, pulling a pen from between his lips and turning to look at Kurt as he said, "Of course it'll be beautiful, Kurt. Let me be very clear about this- I love you and I'm going to try so damn hard to make it absolutely perfect for you. Because I know you will for me. When you're ready, when we're ready, it'll be beautiful." He'd finished sweetly and Kurt had smiled over at him, their eyes searching each other briefly before they turned away and continued with their homework. Both of their heads filled with images of soft sheets and scented candles and a serene quietness and soft moans. I had been wonderful- just imagining it.
Now, though, Blaine looks down at the concrete, down past his scuffed shoes, and he's searching the cracks with wide, worried eyes. Then he looks back over his shoulder and he curses bitterly at the wind. It takes him less than five seconds to spin around and start pounding back down the road, in the direcion he came. Hoping, no, he's outright praying that Kurt hasn't left him. He's praying that his perfect boyfriend is still there waiting for him. Though, he knows he wouldn't blame Kurt for leaving. Not after he walked away like that. Why had he let his emotions take control? Why was he so damn drunk? Why had he let his lust overpower his brain?
We're bigger than all of this, Blaine thinks. Because they are. They're bigger, and far greater, than Ohio and a stupid gay bar and that stupid Warbler who'd been hitting on him all night. They're so much better than all of it. They're so much better.
He's been so stupid. Blaine is practically running now, tears filling his eyes and blinding him as he stumbles over the hill, his eyes search frantically for his boyfriend. His heart only beating again when he realises that Kurt is standing exactly where he left him- but now he looks so compact and so small under the distant yellow-orange glow of a streetlight.
Blaine's heart is practically pounding out of his chest and it's only when he draws closer that he realises Kurt is crying, his shoulders bouncing softly, one of his hands covering his face to hide his distress. The fingers of his free hand stretching outwards- in the general direction Blaine had vanished when he'd ignored Kurt's overwhelming pleas to stay.
Blaine feels sick.
Sick with worry.
Sick with himself.
An uncomfortable lump forms in Blaine's throat, more than big enough to suffocate him, as he walks towards Kurt and hears his boyfriend's tender, repetative whisper, "I'm sorry. I'm so sorry. I'm sorry. I'm so sorry. I'm sorry. I'm so sorry.' and he wants to vanish through the floor. But he wont do that again, he wont go away. Blaine feels like his heart is trying to fall out of his body as Kurt continues to whisper his robotic apologies into the night. It's then that a horrible, heartbreaking moan slips past Kurt's open lips and a purely instinctive need to help, to do something, shoots through Blaine like a spark of electricity. Jolting him into action.
"Kurt-" Blaine whispers finally, reaching out softly to lace his fingers through those outstretched fingertips of his boyfriend. Kurt's head jerks up in shock and for a moment he just stares and stares at Blaine. His eyes painfully red as he looks at him, heartache written so plainly across Kurt's face that Blaine gasps at it's honesty.
"I'm so sorry, Kurt." Blaine mutters then, his words less than certain as his inebriated brain tries to function. He needs to pull himself together. He needs to stop Kurt from hurting, this isn't how it works, they don't hurt each other. There are enough people in the world determined to cause them pain, they don't treat each other like this.
"Y-you came back?" Kurt whispers softly, though, there's no denying the painful shock in his voice and Blaine decides right then that the uncertainty in his voice is possibly more painful than knowing that the tears running down the soft curve of Kurt's face are his doing. It hurts him because he knows that Kurt had thought the worst- he knows Kurt, he knows him inside out, even when he's intoxicated, he knows that Kurt always finds a way to place blame on himself. But this, whatever it is, these tears, they're all Blaine. He's managed to hurt them both.
"I love you." Blaine mutters quietly in reply, desperately trying to explain himself as the light burns at his eyes, "Of course I came back. You're my everything." He stumbles but that just makes Kurt cry harder.
"I'm so s-sorry." Kurt whispers then, as he sniffles into his shirt sleeve, and Blaine doesn't know what he's sorry about- the tears, the fight or the fact that he pushed Blaine away in the back of the car, but he's convinced that Kurt has nothing to apologise for. This was him. This was all him. He shakeshis head.
"No." Blaine says rather firmly then, or at least as firmly as he can manage as his head pounds, "Don't do that. Don't you start apologising for something that I did. God, Kurt. I'm so sorry. I just.. I'm so drunk right now and you're so beautiful, you're so damn sexy and I wasn't.. I wasn't thinking straight." Blaine's the one pleading now.
"I-it's okay." Kurt mutters quickly and Blaine grabs at his boyfriend's hands, swaying a little as he stares at him through a blurry haze, "No, it's not. It's not okay. I want… I want our first time to be perfect, Kurt. As perfect as you are. I want us to be somewhere safe, I want us to be at home - in private - with a soft bed beneath us and flickering candles around us and those sweet scented oils burning away. I don't.. I don't want to cheapen that experience for you, for us, because… because that means I'm cheapening you too and, Jesus, Kurt, you don't deserve that. You'll never deserve that, not from me.. not from anyone. Okay? Shit, look. Kurt, I'm just so sorry. I'm so fucking sorry… and if I made you feel dirty or if.. if you felt pressured. Oh, God, if you felt pressured. I don't- oh, fuck- I pressured you, Kurt. Shit. You have every right to say no to me, Kurt Hummel. Every fucking right. Always. Okay? I never want to make you feel trapped or… or powerless. I don't want to hurt you. Oh, god, I've screwed up so bad. So fucking bad. Shit. Kurt-" It's then that Kurt tugs on Blaine's hands to grab his attention and, when he does look up, something has changed. The sadness in Kurt's face has shifted. There's something else there now. Something that looks a lot like concern.
"You didn't pressure me, okay? Don't think that you did." Kurt says when Blaine's eyes meet his, the firmness in his voice and his posture steadily returning, "I never felt trapped, not for a single second, and you didn't hurt me, Blaine. Okay? I know you care about me, that you love me, and that you'd always stop if I asked you to. You didn't hurt me, Blaine…. at least… not until you... not until you walked away from me." Kurt's expression falters dramatically.
"Oh, God, I promise you.. I'll never walk away. Never again Kurt. Never." Blaine promises quickly, stumbling over his words as his fingers squeeze Kurt's fiercely.
There's a moment of silence then.
A pause where they both take a soundless moment to understand each other.
Then it's gone.
Kurt pulls a hand away from Blaine and nudges him playfully in the arm, "You couldn't walk away if you tried. You smell like a brewery, sweetheart." Kurt whispers teasingly, a ghost of a smile tugging at the corners of his mouth.
He's almost okay again and Blaine can almost breathe.
"Kurt? Can we just go home, please?" Blaine asks quietly, almost shyly, and Kurt nods once in understanding, tilting his head a little before he opens the door and helps Blaine climb inside.
This time, when Kurt leans across to fasten his boyfriend's seatbelt Blaine doesn't drag him inside, not this time, this time he just leans forward and knocks their heads together before he presses a gentle kiss against Kurt's temple.
"I love you, Kurt." Blaine mutters sincerely, warm against the shell of his boyfriend's ear, and Kurt just smiles in return, leaving a soft kiss in Blaine's unruly hair as he moves away. Whispering into the quietness, "I know you do, sweetheart. I love you, too." Then Kurt's hand moves to cup one of Blaine's cheeks - so he can wipe away the shimmering tear tracks that stain his face. Blaine hadn't even noticed his own tears.
Kurt gives his boyfriend one last lingering look, before he closes the door and whispers into the night, "I couldn't let tonight happen,Blaine, I want you to remember how happy we are together. We both deserve that much."


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Cars are sold on progress. New models! New features! New cupholders! Sometimes, though, there are cars that remain timelessly desirable.
Much as I respect the 691 horses bottled up in the Aventador's V12, there's no new Lambo that I would want more than a Miura. This one's an SV, the most lust-worthy of all.


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Nephritis is the common kidney disease. In clinic there are many symptoms such as protein urine, blood urine, edema,high blood pressure and so on. Among of them, the protein urine refers to the ultra immune response on the kidney and the in...Read More
For patients with nephritis and high blood pressure, they may have such a doubt: what is the proper diet for me? Then let us have a look at the following contents. In normal condition, kidneys can filter metabolic products and extra fluids...Read More
Nephritis refers to the inflammation of glomeruli in the kidneys. For patients with this disorder, a proper diet can help delay its progression to a large degree. Then what is the healthy diet plan? ...Read More
Name: MADRID Gender: Female Age : 68 Country: Honduras Illness condition : diabet...Read More
Her name is naher and her is from Bangladesh. Since she didnt receive a effective...Read More
Name : Haasan Gender : male Age :62 Country : Oman Illness Condition : Diabetes a...Read More
US Contact:+86-15233618520


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Bolle Helix Sunglass Replacement Lenses, Modulator Rose
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Search our site to find another great deal. But hurry! With our low prices, we sell out fast!
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- Features
- Bolle Helix replacement lens keeps you in the action no matter what conditions are thrown in your way. Helix sunglasses lenses from bolle are extremely useful sunglasses accessories, but with the level of strength and durability that bolle eyewear accessories are known for. Bolle Helix interchangeable lenses are essential for keeping up with the action during changing weather conditions or drastic changes in light. There is no better sunglasses accessory for your bolle Helix sun glasses than the Helix replacement lenses! bolle interchangeable Helix lens is part of the bolle performance Series, making no compromises to achieve maximum performance.
- 054917274276
- Brand
- Bolle
- Model
- 50268
- Part
- 50268
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