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The macrophage is the primary host cell for the fungal pathogen Histoplasma capsulatum during mammalian infections, yet little is known about fungal genes required for intracellular replication in the host. Since the ability to scavenge iron from the host is important for the virulence of most pathogens, we investigated the role of iron acquisition in H. capsulatum pathogenesis. H. capsulatum acquires iron through the action of ferric reductases and the production of siderophores, but the genes responsible for these activities and their role in virulence have not been determined. We identified a discrete set of co-regulated genes whose transcription is induced under low iron conditions. These genes all appeared to be involved in the synthesis, secretion, and utilization of siderophores. Surprisingly, the majority of these transcriptionally co-regulated genes were found clustered adjacent to each other in the genome of the three sequenced strains of H. capsulatum, suggesting that their proximity might foster coordinate gene regulation. Additionally, we identified a consensus sequence in the promoters of all of these genes that may contribute to iron-regulated gene expression. The gene set included L-ornithine monooxygenase (SID1), the enzyme that catalyzes the first committed step in siderophore production in other fungi. Disruption of SID1 by allelic replacement resulted in poor growth under low iron conditions, as well as a loss of siderophore production. Strains deficient in SID1 showed a significant growth defect in murine bone-marrow-derived macrophages and attenuation in the mouse model of infection. These data indicated that H. capsulatum utilizes siderophores in addition to other iron acquisition mechanisms for optimal growth during infection.
Author Summary
Fungal infections are a growing public health threat, particularly for immunocompromised individuals such as people with AIDS, organ transplant recipients, and cancer patients. Present antifungal therapies are often highly toxic and resistance to these therapies continues to rise. Histoplasma capsulatum is a pathogenic fungus that infects humans, causing pulmonary and systemic disease. It is the most common cause of fungal respiratory infection in the world, and is endemic to the Mississippi and Ohio River valleys of the United States. H. capsulatum produces small molecules, called siderophores, to acquire iron, an essential nutrient. We have identified genes that are involved in the synthesis of siderophores in this fungus and have found that siderophore production in H. capsulatum is important for its virulence. Since siderophore production is confined to microbes and plays no role in human biology, it is an excellent target for rational drug design.
Citation: Hwang LH, Mayfield JA, Rine J, Sil A (2008) Histoplasma Requires SID1, a Member of an Iron-Regulated Siderophore Gene Cluster, for Host Colonization. PLoS Pathog 4(4): e1000044. https://doi.org/10.1371/journal.ppat.1000044
Editor: Alex Andrianopoulos, University of Melbourne, Australia
Received: November 2, 2007; Accepted: March 13, 2008; Published: April 11, 2008
Copyright: © 2008 Hwang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by The Leukemia & Lymphoma Society Special Fellow Award 3107-03 to LH, NIH HG00047 and Damon Runyon Cancer Research Fund to JM, NIH GM31105 and GM35827 to JR, NIH (RO1AI066224, PO1AI063302, and UO1AI150934) to AS, the Sandler Program in Basic Sciences to AS, and the Howard Hughes Medical Institute Biomedical Research Support Program Grant (5300246) to the UCSF School of Medicine.
Competing interests: The authors have declared that no competing interests exist.
Iron acquisition is critical to cellular function and survival. During infection of mammals, the host limits the access of iron to microbial pathogens by a variety of means [1]. In turn, pathogens utilize a number of strategies to acquire iron in the face of iron restriction by the host. Here we investigate the role of siderophore-mediated iron acquisition in the fungal pathogen Histoplasma capsulatum, which parasitizes host macrophages during infection.
Histoplasma capsulatum is a dimorphic, fungal pathogen that causes respiratory and systemic disease in humans. Infection of mammals is initiated by inhalation of fungal spores from soil in regions of the U.S. where the organism is endemic. Once in the host, H. capsulatum grows in a budding yeast form that colonizes alveolar macrophages. Yeast cells replicate within the macrophage phagolysosome, but the molecular mechanisms governing survival within host cells remain largely undefined [2].
Replication of H. capsulatum within macrophages is dependent on the availability of iron. During infection of murine peritoneal and human monocyte-derived macrophages, addition of the iron chelator deferoxamine inhibits the intracellular growth of H. capsulatum. This inhibition is suppressed by the addition of iron-rich transferrin (holotransferrin), indicating that active iron acquisition is a critical determinant of intracellular growth [3],[4]. In addition, the adaptive immune response to H. capsulatum infection, which triggers production of the cytokine interferon-gamma (IFNγ) by T-cells [5], may curtail intracellular fungal growth by limiting iron acquisition by the fungus. Treatment of murine peritoneal macrophages with interferon-gamma (IFNg) causes growth restriction of H. capsulatum which, in turn, can be reversed by addition of holotransferrin [3]. IFNγ downregulates surface transferrin receptors, suggesting that a major means by which IFNγ inhibits intracellular fungal growth is via iron limitation.
These studies indicate that iron acquisition plays a critical role in Histoplasma virulence. However, although several biochemical activities have been identified in H. capsulatum, little is known about the genes that regulate iron accumulation. H. capsulatum is known to secrete hydroxamate siderophores that act as low-molecular-weight ferric iron chelators under low iron conditions [6],[7]. Additionally, iron limitation also induces reductive iron assimilation, including ferric reductase activity [8].
To assess the role of these iron acquisition mechanisms in virulence, we identified H. capsulatum genes that function in siderophore-mediated iron acquisition. These genes were transcriptionally induced under low iron conditions and contained a common putative regulatory site in their upstream regions that might govern their coordinate expression. Inspection of the sequences revealed that these genes were clustered together in the genome, and thus defined a secondary metabolite gene cluster involved in siderophore biosynthesis. Disruption of one of the genes of this pathway, SID1, resulted in elimination of hydroxamate siderophore synthesis, diminished growth of H. capsulatum within macrophages, and compromised virulence in mice. These data indicated that iron scavenging though siderophore production facilitates the parasitic growth of H. capsulatum.
Transcriptional response to low iron
The genes involved in iron acquisition are tightly regulated at the level of transcription in most microorganisms. To identify genes that are transcriptionally regulated by iron limitation conditions in H. capsulatum, we grew H. capsulatum (strain G217B) yeast cells under iron-limiting (100 µM deferoxamine mesylate) or iron-replete (5 or 10 µM FeSO4) conditions for various periods of time. The transcriptional profiles of these samples were compared using whole-genome oligonucleotide microarrays. We found seven genes that were transcriptionally induced under conditions of iron limitation and named them according to the putative functions of the corresponding proteins (Figure 1A). These genes included L-ornithine monooxygenase (SID1, previously named LOM1 [9], EU253976), the enzyme catalyzing the first committed step in extracellular and intracellular siderophore production in other organisms; an acetylase (SID3, EU253977); an acid co-A ligase (SID4, EU253978); a non-ribosomal peptide synthase (NPS1, EU253973); an oxidoreductase (OXR1, EU253974); a major facilitator superfamily (MFS) transporter (MFS1, EU253970) [10]; and an ATP-binding cassette transporter (ABC1, EU253969) [10].
A. Iron-dependent gene expression cluster from whole genome oligonucleotide microarray analysis of G217B yeast grown under iron replete (5 µM FeSO4) and iron limited (100 µM deferoxamine mesylate) conditions for 0, 4, 24, and 48 hours, as described in Materials and Methods. Red indicates up-regulated genes, green indicates down-regulated genes, black indicates genes that do not change in expression, and gray indicates that no data are available. Microarray time courses were repeated with three biological replicates. A representative experiment is shown. Similar results were seen for iron replete conditions of either 5 or 10 µM FeSO4. B. Quantitative RT-PCR of siderophore biosynthesis and utilization genes. Total RNA from the 0, 1, 4, and 24 hr time points used in the microarray analysis were analyzed for relative transcript levels using QRT-PCR. Transcript levels were normalized to ACT1 transcript. Relative quantities were normalized to t = 0 values (black bars). For the 1, 4, and 24 hr time points, white bars indicate growth in medium supplemented with FeSO4 (+Fe), and gray bars indicate growth in deferoxamine (+Df).
Based on precedent from other fungi, these genes were likely to have roles in the production, transport, and utilization of siderophores, and could comprise the entire pathway of hydroxamate siderophore production [11]–[13]. SID3 is orthologous to SidF in Aspergillus fumigatus, an N5-transacylase involved in the synthesis of fusarinine and triacetylfusarinine [13]. NPS1 is orthologous to the non-ribosomal peptide synthases involved in extracellular siderophore production in ascomycetes [13],[14]. MFS1 is homologous to mirB of Aspergillus nidulans, a siderophore transporter [15]. And finally, ABC1 contains homology to MTABC3, a mammalian ABC transporter involved in iron homeostatis [16].
Quantitative RT-PCR (QRT-PCR) analysis confirmed that all seven genes were transcriptionally induced during iron deprivation (Figure 1B). Both the microarrays and QRT-PCR analysis also showed that transcription of these genes began to be induced even in the presence of iron supplementation at late time points, suggesting that iron eventually becomes limiting under these conditions, or perhaps that the induction of genes involved in siderophore synthesis is highly sensitive to a subtle decrease in iron availability.
Surprisingly, all but one of these iron-regulated genes were located adjacent to each other in an approximately 25 kb region of the genome of the G217B strain of H. capsulatum (Figure 2A). Only MFS1 is encoded by a distinct genomic region. This genomic cluster of iron-regulated genes was also present in the two other sequenced strains of H. capsulatum: G186AR (Genome Sequencing Center, Washington University, St. Louis, MO) and WU24 (Broad Institute, Massachusetts Institute of Technology, Cambridge, MA). In G186AR, an additional MFS transporter, MFS2 (EU253971), as well as a second acetylase (SID5, EU253979), was found within the putative iron-regulated gene cluster. WU24 contained the 5′ end of the MFS2 gene, but did not contain SID5. We observed a similar genome structure in the closely related fungus Coccidioides immitis (data not shown), but only a subset of the genes were found adjacent to each other in other fungal genomes (see discussion).
A. Schematic showing genome structure of siderophore biosynthesis and utilization genes in H. capsulatum strains G217B, G186AR, and WU24. B. Location and sequence of putative consensus site in promoters of siderophore genes in the G217B strain. One kilobase of sequence 5′ to the open reading frame for each gene is represented by a black line, except for the divergent genes: SID4 and SID1, with an intergenic region of 1036 bases; RTA1 and NIT22, with an intergenic region of 2762 bases; and NPS1 and ABC1, with an intergenic region of 1677 bases. The location of the consensus sequence is marked with a short vertical line and the associated number represents the number of base pairs from the start codon of the downstream gene. The 5′ to 3′ orientation of the consensus site is not indicated.
Two of the genes located within this genomic cluster, NIT22 (encoding a dehydratase, [9], EU253972) and RTA1 (encoding a predicted protein containing an RTA domain, resistance to 7-aminocholesterol, EU253975), did not appear to be iron regulated by microarray. We used QRT-PCR analysis to determine whether these genes were regulated differentially in response to iron levels. However, whereas RTA1 expression was not detected by QRT-PCR under either iron-rich or iron-poor conditions (data not shown), NIT22 expression was induced under iron limitation (Figure 1B). Unlike the other iron-regulated genes in this genomic cluster, NIT22 did not encode a protein with homology to known siderophore-biosynthesis genes. Thus, its induction under conditions of iron limitation may reflect a previously unidentified role of its dehydratase activity in siderophore biosynthesis. To determine if the iron-dependent regulation of this gene cluster extended beyond the genes we identified by microarray, we also examined the expression of UBP1 (encoding a ubiquitin C-terminal hydrolase), a gene approximately 6kb upstream of OXR1. UBP1 expression was not regulated by iron levels (Figure 1B). In addition, we used QRT-PCR to determine that genes found in the iron cluster of G186AR were also transcriptionally regulated by iron levels (Figure S1).
Identification of a putative regulatory site
Since the genes in this siderophore biosynthetic cluster were transcriptionally co-regulated in response to iron levels, we used Multiple Em for Motif Elicitation (MEME) to identify conserved sequences in the upstream regions of these genes from both the G217B and G186AR strains of H. capsulatum. A consensus site, 5′-(G/A)ATC(T/A)GATAA-3′, was present at least once in the 5′ regions of all of the genes in the siderophore biosynthetic cluster (Figure 2B). Interestingly, this consensus contained an HGATAR sequence, the recognition site for fungal GATA transcriptional regulators [17], some of which are known to regulate the expression of siderophore-biosynthesis genes in other fungi [18]–[20]. In H. capsulatum, an ortholog of these GATA factors, Sre1, was shown to bind in vitro to the consensus site we identified here, suggesting that Sre1 limits expression of the siderophore biosynthetic gene cluster under iron-replete conditions (Chao et al, submitted).
Disruption of SID1 eliminated siderophore production
To test whether this pathway was important for siderophore production, we disrupted SID1, which encodes the enzyme that catalyzes the first committed step in siderophore production. The gene disruption was performed in the wild-type G186ARura5Δ strain background and confirmed by PCR (data not shown) and Southern blot analysis (Figure 3A). The sid1Δ strain grew with normal kinetics under iron-replete conditions, but displayed a growth defect under conditions of iron limitation (Figure 3B). Consistent with its poor growth under iron-limiting conditions, the sid1Δ strain also failed to produce siderophores (Figure 3C).
A. Southern analysis of SID1 disruption. Genomic DNA from parental strain (WU8-Wild-type) and potential sid1Δ::hph strains (HcLH25, HcLH26, and HcLH27) was digested by EcoRI or SalI, separated by gel electrophoresis, blotted and hybridized to probes containing either the open reading frame of SID1 or the hph gene. For the SID1 probe, fragments of the expected sizes of 6.4 kb (EcoRI) and 12.2 kb (SalI) are seen in the DNA from the parental strain. For the hph probe, fragments of the expected sizes of 3.6 kb and 2.3 kb (EcoRI) and 6.8 kb (SalI) are seen in the DNA from sid1Δ strains. B. Growth curves of HcLH95 (WT+vector), HcLH97 (sid1Δ+vector), HcLH103 (WT+SID1), and HcLH106 (sid1Δ+SID1). Yeast cells were grown in mRPMI medium (pH 6.5) without or with 5 µM FeSO4 added (+Fe). Growth and siderophore production experiments were repeated at least three times with two independent sid1Δ strains and four independent complementation strains. Samples for OD600 were taken in triplicate. The mean+/−standard deviation is indicated. A representative experiment is presented. C. Siderophore production of strains. Yeast cells (same strains used in B) were grown in mRPMI medium (pH 6.5) for 24 hours and culture supernatant was examined for presence of siderophores using chrome azurol S assay as described in Materials and Methods. Samples were taken in triplicate. The mean+/−standard deviation is indicated. Results are given as percentage of siderophore production compared with WT+vector.
To insure that the observed growth defect was due to disruption of SID1, we complemented the sid1Δ strain with a wild-type copy of the SID1 gene. SID1, including all intergenic regions 5′ and 3′ of the open reading frame, was cloned into an integrating Agrobacterium tumefaciens T-DNA vector (pED2). This construct was randomly integrated into the wild-type genome and into two independent sid1Δ isolates. The SID1 gene fully complemented the growth defect in low-iron medium (Figure 3B), but only partially complemented siderophore production (Figure 3C) for unknown reasons. QRT-PCR analysis revealed that SID1 transcript accumulation was similar under iron-limiting conditions in the wild-type and complemented strains (data not shown). We also introduced a SID1 complementation construct on a high-copy episomal plasmid, but these transformants expressed SID1 at very low levels and showed little complementation of siderophore production (data not shown), suggesting that integration into the genome may be required for normal levels of SID1 transcription.
SID1 is important for virulence
In other pathogens, iron acquisition from the host is a critical virulence determinant. To examine the role of siderophore-mediated iron acquisition in H. capsulatum infection, we infected bone-marrow-derived murine macrophages (BMDMs) with wild-type, sid1Δ, and complemented strains. At zero, 6, 24, and 48 hours following infection, the macrophages were lysed and colony-forming units (CFUs) of H. capsulatum cells were determined (Figure 4A). Wild-type H. capsulatum cells were able to proliferate within the macrophages as expected. Although the sid1Δ strains were also able to grow intracellularly, they reached only ∼40% of wild-type levels by 48 hours after infection (p<0.001). The doubling time of wild-type cells in macrophages was 10 hours while the sid1Δ strain doubled in 14 hours. Addition of 100 µM FeSO4 to infected macrophages reversed this phenotype, strongly suggesting that the proliferation defect in macrophages was due to the decreased ability of the mutant strains to acquire iron (Figure 4B). The complemented strains did not display a growth defect in macrophages, indicating that they made sufficient siderophores to permit wild-type growth during macrophage infection (Figure 4A). The growth defect of the mutant strain and complementation of the defect with either reconstitution of the wild-type gene or the addition of iron was consistent over several infections (Figure 4C).
HcLH95 (WT+vector), HcLH97 (sid1Δ+vector), HcLH103 (WT+SID1), and HcLH106 (sid1Δ+SID1) were used to infect C57BL/6J bone-marrow-derived macrophages at a multiplicity of infection of 5. Macrophages were lysed at t = 0, 6, 24, and 48 hours, and plated for H. capsulatum colony-forming units (CFUs) on HMM. CFUs were normalized to the zero time point. Infected macrophages were incubated in either BMM (A) or BMM+100 µM FeSO4 (B) Data from a representative time course is shown. The average of three platings+/−standard error of the mean is shown. Significance was determined using ANOVA with Bonferroni Multiple Comparisons Test. Comparison of wt+vector and sid1Δ+vector at 48 hours has a p<0.001. The experiment was repeated three times with two independently isolated sid1Δ strains, as well as four independently isolated sid1Δ+SID1 strains. (C) The average CFU at 48 hours of three independent infections+/−standard error of the mean is shown. White bars indicate CFUs from infected macrophages incubated in BMM, grey bars indicate macrophages incubated in BMM+100 µM FeSO4. Significance was determined for the influence of genotype (WT versus sid1Δ) and iron using three-way ANOVA. Genotype influence, *: p-value<0.05.
To investigate whether siderophore production plays a role in animal infections, we first infected C57BL/6J mice intranasally with a lethal dose (effective inoculum of ∼1×105 CFU) of either wild-type, sid1Δ, or complemented strains. No significant differences in pulmonary fungal burden were observed under these conditions (data not shown). However, since the literature suggested that assessing the virulence of a mutant strain in competition with wild-type can provide a more sensitive assay for pathogenesis defects [21],[22], we decided to perform a competitive infection with wild-type, sid1Δ, and the complemented strains. We infected C57BL/6J mice intranasally with a sublethal dose (effective inoculum of ∼1×104 CFU) of an equal mix of either wild-type and sid1Δ or wild-type and sid1Δ+SID1 strains. Lung homogenates from multiple time points were analyzed for CFUs and ratios of wild-type, sid1Δ, and complemented strains were calculated. The competitive index, which reflects the defect of a particular strain with respect to the wild-type strain, was determined as described in Materials and Methods. We observed that the sid1Δ strain showed a significant defect in pulmonary colonization compared to wild-type cells (Figure 5). This defect began at day 5, with a severe defect apparent at day 15 (p<0.01). This phenotype was reversed in the complemented strain, which accumulated to slightly higher levels than the wild-type strain for unknown reasons (p<0.05). These data indicated that siderophore production is important for optimal growth of H. capsulatum in mouse lungs.
C57BL/6J mice were infected intranasally with an effective inoculum of 1×104 CFU of an equal mix of hcLH95 and hcLH97 (wt:sid1Δ, black boxes) or hcLH95 and hcLH106 (wt: sid1Δ+SID1, white boxes). Boxes indicate the competitive index (CI) for individual mice as determined in Materials and Methods. Horizontal bars indicate the means of CI values. A pairwise comparison of the 15-day CI for the wt:sid1Δ and wt: sid1Δ+SID1 infections by the Mann-Whitney/Wilcoxon test revealed that they were significantly different (p = 0.0286). Significance for WT:sid1Δ time course was determined using Kruskal-Wallis test (ANOVA) with Dunn's Multiple Comparisons test. This analysis showed that the CI at t = 4 hr and t = 15 days was significantly different (p<0.01).
Iron acquisition is essential for the growth of most microorganisms and occurs primarily by two mechanisms: siderophore production and reductive iron assimilation [23]. Both of these processes are induced by H. capsulatum during iron limitation [6],[8], but their role in cell growth and virulence has not been previously investigated. In this study, we determined the role of siderophore production in growth under conditions of iron limitation as well as during infection. We identified a siderophore biosynthetic gene cluster that was transcriptionally induced in response to iron limitation. Disruption of siderophore production resulted in iron-dependent growth in culture and during macrophage infection, and caused a growth defect in mice.
Presumably the sid1Δ mutant shows a strong pulmonary colonization defect in competition with wild-type because it has a reduced capacity for iron acquisition in vivo. The in vivo growth defect caused by lack of siderophore production is most pronounced at 15 days post infection. Interestingly, the peak of IFN-γ production by T cells in response to H. capsulatum infection occurs at day 14 [24]. Since one of the functions of IFN-γ is to restrict iron, perhaps siderophore production is primarily required for iron acquisition during the latter stages of infection, whereas redundant iron acquisition mechanisms, such as reductive iron assimilation, might play a role early in infection.
In other fungal pathogens, siderophore production and reductive iron assimilation, which is usually mediated by ferric reductase activity, play differential roles in pathogenesis. For example, siderophore production is essential for virulence in Aspergillus fumigatus, but reductive iron assimilation is neither necessary nor sufficient for normal growth and survival in the host [25]. Similarly, in the phytopathogens Cochliobolus miyabeanus, Alternaria brassicicola, Cochliobolus heterostrophus, and Fusarium graminearum, extracellular siderophore production is required for full virulence [14]. In contrast, deletion of the SID1 gene in U. maydis does not affect virulence in maize [26]; instead, reductive iron assimilation is required for virulence [27]. This also true in Candida albicans, where ferric reductase activity is required for systemic infection [25],[28].
Ferric reductase activity has been clearly demonstrated in H. capsulatum [8], though the relevant genes that encode these activities have not been identified, and thus their role in virulence has not been assessed. Perusal of the genome revealed seven putative ferric reductase genes, though none of these candidates were observed to be upregulated by iron limitation in our experiments. Nonetheless, any or all of these genes could contribute to pathogenesis in the host.
In addition to identifying a role for siderophore production in virulence, this study revealed several interesting regulatory properties of siderophore-biosynthesis genes. First, inspection of the sequences upstream of each gene revealed a consensus sequence that was present at least once per gene. The expression of several fungal orthologs of SID1 is repressed by GATA-type negative regulators that recognize the HGATR motif [19],[20],[29]. Interestingly, the consensus sequence we identified, 5′-(G/A)ATC(T/A)GATAA-3′, contained an HGATAR motif, and was shown to bind an H. capsulatum GATA factor in vitro (Chao et al., submitted). We are now investigating whether these regulatory sequences are necessary to confer gene regulation in response to iron levels in vivo.
Second, the siderophore biosynthesis genes were located adjacent to each other in the genome, and thus comprise the first secondary metabolite gene cluster defined in H. capsulatum. We observed similar clustering in the genome of the closely related systemic dimorphic fungal pathogen C. immitis, but it is not evident to nearly the same extent for siderophore biosynthesis genes in other sequenced fungal genomes [27],[30],[31].
Third, we could not express significant levels of SID1 from an episomal plasmid, suggesting that integration of the gene may be critical for normal expression levels. Additionally, integration of SID1 at random sites in complementation strains resulted in partial complementation of siderophore production, suggesting that optimal function might be achieved only with expression from the original genomic locus. Unfortunately, targeted integration is extremely challenging in H. capsulatum, making it difficult to directly test this hypothesis.
The potential significance of the H. capsulatum gene cluster with regards to gene regulation is intriguing. One possibility is that clustering in the genome facilitates local changes in chromatin structure that allow a regional change in promoter accessibility. This type of regulation is reminiscent of transcriptional control of secondary metabolite clusters in Aspergillus species, where transcriptional accessibility of genes in the cluster is controlled by the putative methyltransferase LaeA [32],[33]. Perhaps local control of chromatin structure allows a rapid and coordinated transcriptional switch in response to changes in iron levels.
Materials and Methods
Strains and culture conditions
Histoplasma capsulatum strains G217B (ATCC 26032) and G186ARura5Δ (WU8), all kind gifts from the laboratory of William Goldman, Washington University, St. Louis, as well as strains generated in this study (Table 1) were grown in HMM broth or plates [34], or in mRPMI broth [RPMI 1640 medium without phenol red or bicarbonate (Invitrogen, www.invitrogen.com), supplemented with 1.8% dextrose (Fisher Scientific, www.fishersci.com), 25 mM HEPES pH 6.5 or pH 7.5 (Fisher Scientific), and 100 units/mL of both penicillin and streptomycin (UCSF Cell Culture Facility, www.ccf.ucsf.edu), modified from [8]]. Unlike HMM, mRPMI contained no added iron, but did not eliminate trace amounts of iron, though cultures grown in mRPMI medium were incubated in plastic flasks to reduce trace iron contamination. Media was supplemented with various concentrations of FeSO4 (Fisher Scientific) as described in the text. HMM and mRPMI media were supplemented when needed with 200 µg/mL hygromycin (Roche, www.roche.com) and 200 µg/mL uracil (Sigma-Aldrich, www.sigmaaldrich.com). Cultures were grown at 37°C under 5% CO2.
For microarray studies, an initial culture of G217B yeast cells was grown in 5 mL HMM, and then passaged 1∶25 into 80 mL HMM. After 3 days of growth, the culture was pelleted, washed in 80 mL of PBS, and resuspended in 1 L of mRPMI pH 7.5 supplemented with 5 µM FeSO4. After 24 hours of growth, 200 mL of culture was harvested for the zero time point. Then the culture was split into 2×400 mL, and an additional 5 µM or 10 µM FeSO4 was added to one culture and 100 µM deferoxamine mesylate (Sigma-Aldrich) was added to the other. At each time point, 100 mL of culture was harvested and processed as below.
For growth, siderophore production, and QRT-PCR assays in G186AR based strains, cells were grown in 5 mL HMM. When the cultures reached late log phase, they were sonicated twice for 3 seconds to disperse cells and then passaged 1∶25 into HMM. After 24 hours of growth, they were pelleted, washed in an equal volume of PBS, and resuspended in an equal volume of mRPMI pH 6.5. For growth curves, triplicate samples of 1 mL of cells were taken at each time point, sonicated twice for 3 seconds, and dilutions were measured by spectrophotometer at OD600. For QRT-PCR, after resuspension in mRPMI pH 6.5, cells were grown for an additional 24 hours. At that point, a t = 0 sample was harvested, then the culture was split. One culture was maintained in mRPMI pH 6.5 media with no additions, the other was treated with 5 µM FeSO4. Cells were harvested at 1, 4, and 24 hours and total RNA was isolated using a guanidine thiocyanate lysis protocol as previously described [10].
Electrotransformation of DNA
Approximately 100 ng of PacI-linearized plasmid DNA with exposed telomere ends was transformed into yeast cells as previously described [35].
Agrobacterium Transformation
H. capsulatum yeast cells were transformed using Agrobacterium-mediated gene transfer as described previously [36]. Briefly, the A. tumefaciens strain (LBA1100, a kind gift of Thomas Sullivan and Bruce Klein with permission from Paul Hooykas (Leiden University, Leiden, The Netherlands)) transformed with the desired plasmid was induced overnight with 200 µM acetosyringone (AS, from Sigma-Aldrich). H. capsulatum yeast cells were harvested from 4 day patches on HMM+uracil plates and diluted to 5×108 cells/mL. Equal volumes of the H. capsulatum and A. tumefaciens cultures were mixed, and 400 µL of the mix was spread onto BiodyneA nylon membranes (Pall Gelman, www.pall.com) on IM agarose plates containing 200 µM AS and 200 µg/mL uracil. Co-cultivation plates were incubated at 28°C for 3 days. The membranes were then transferred onto HMM plates with no added uracil and incubated at 37°C for 2 to 3 weeks. Strains WU8, HcLH25, and HcLH26 were transformed with pVN61 (vector control marked with URA5) and pED2 (SID1 complementation marked with URA5). All of the transformed strains show similar in vitro growth in liquid and solid media without uracil.
Cultures of H. capsulatum were harvested by filtration, and total RNA was isolated using a guanidine thiocyanate lysis protocol as previously described [10]. This RNA was used for both microarrays as well as QRT-PCR. For microarrays, polyadenylated RNA was purified from total RNA using an Oligotex mRNA kit (Qiagen Inc.,www.qiagen.com). cDNA synthesis from polyA-selected RNA and fluorescent labeling was performed as described previously [37]. Briefly, an equal mass of RNA from each time point was pooled to generate a reference sample and labeled with Cy3. cDNA from each individual time point was labeled with Cy5 and competitively hybridized versus the reference pool using H. capsulatum G217B 70-mer oligonucleotide microarrays. The microarrays contain a single 70-mer oligo for each predicted gene in the G217B genome, as well as two 70-mer oligos for low confidence genes.
Arrays were scanned on a GenePix 4000B scanner (Axon Instruments/Molecular Devices, www.moleculardevices.com) and analyzed using GENEPIX PRO, version 6.0 (Molecular Devices), Spotreader (Niles Scientific, www.nilesscientific.com), NOMAD 2.0 (http://nomad2.ucsf.edu/NOMAD/nomad-cgi/login.pl), CLUSTER [38], and Java Treeview 1.0.12 (available at http://sourceforge.net/project/showfiles.phpgroup_id84593). To eliminate elements with low signal, we did not analyze elements for which the sum of the medians for the 635-nm and 532-nm channels was ≤500 intensity units. All of the time points were normalized relative to the zero time point.
Quantitative RT-PCR
Total RNA was treated with DNaseI (Promega, www.promega.com). cDNA was synthesized from 3.3 µg of DNaseI-treated RNA using Stratascript reverse transcriptase (Stratagene, www.stratagene.com) and oligo-dT. Quantitative PCR was performed on 1∶100 dilutions of cDNA, except for MFS2 and SID5 reactions, which used 1∶40 dilutions of cDNA. The reactions included 1.5 mM MgCl2, 1× Amplitaq buffer, 0.6 units Amplitaq Gold (Applied Biosystems, www.appliedbiosystems.com), 1× SYBR Green (Molecular Probes, probes.invitrogen.com), and 200 nM primers. Reactions were performed on the Mx3000P QPCR system (Stratagene, www.stratagene.com) with Comparative Quantitation (with dissociation curve) program, using actin (ACT1) as the normalizing transcript. Cycling parameters were 95°C for 10 minutes, then 40 cycles of 95°C (30 s), 57°C (1 min), 72°C (30 s) followed by dissociation curve analysis. All reactions were performed in triplicate. A calibrator sample made from an equal mix of each RNA sample was included on each plate. Reactions were analyzed using MxPro software. Primer sequences are included in supplemental material (Table S1).
Accession Numbers
The H. capsulatum GenBank/NCBI (http://www.ncbi.nlm.nih.gov/) nucleotide sequences for the genetic loci described in this publication are ABC1 (EU253969), MFS1 (EU253970), MFS2 (EU253971), NIT22 (EU253972), NPS1 (EU253973), OXR1 (EU253974), RTA1 (EU253975), SID1 (EU253976), SID3 (EU253977), SID4 (EU253978), and SID5 (EU253979).
Consensus site identification
Promoter regions (including 1 kb upstream of ATG, or entire intergenic regions for SID4/SID1 and NPS1/ABC1) of SID3, SID4, SID1, NIT22, NPS1, ABC1, OXR1, and MFS1 from G217B and G186AR were submitted for MEME (Multiple Em for Motif Elicitation) analysis at http://meme.sdsc.edu. The length range for consensus site was set between 6 and 50 base pairs.
Disruption of SID1
A positive-negative selection strategy was utilized to disrupt SID1 in G186ARura5Δ (WU15), as described in [39]. Originally, we attempted to disrupt this gene in the G217Bura5-23 strain, but we were unsuccessful. A disruption construct, pLH36, was made containing 1213 bps 5′ and 943 bps 3′ of the SID1 open reading from G217B. The open reading frame was replaced with the hygromycin resistance gene, hph under the control of the Aspergillus nidulans gpd promoter. This construct was introduced into G186ARura5Δ (WU15) by electrotransformation. Seven independent transformants were colony purified, inoculated into HMM+hygromycin+uracil+20 µM FeSO4 medium and passaged 3 times (1∶25 dilution) once the cultures had reached mid log phase. After the final passage, serial dilutions of the cells were plated onto HMM (pH 4.5)+1 g/L 5-Fluoroorotic acid (5-FOA, Zymo Research, www.zymoresearch.com) +20 µM FeSO4 agarose plates. Genomic DNA from colonies on 5-FOA plates was tested by PCR for the disruption. Potential gene disruptions were further tested by Southern analysis.
Quantification of siderophores
Secretion of siderophores was detected using chrome azurol S (CAS) [40]. Briefly, 0.5 mL of culture supernatant was mixed with 0.5 mL of CAS assay solution (600 µM hexadecyltrimethyl ammonium (Sigma Aldrich), 15 µM FeCl3 (Sigma Aldrich), 150 µM CAS (Sigma Aldrich), 500 mM anhydrous piperazine (Fluka), 750 mM HCl (Fisher Scientific), and 4 mM 5-sulfosalicylic acid (Sigma Aldrich)), incubated for 1 hour, and the OD630 was measured with a spectrophotometer.
Macrophage culture and infections
Bone-marrow-derived macrophages (BMDM) were isolated from femurs of 6- to 8-week old female C57BL/6J mice. The bone marrow was eluted using BMM (Bone Marrow Macrophage) medium, which consists of Dulbecco's Modified Eagle Medium, D-MEM High Glucose (UCSF Cell Culture Facility), 20% Fetal Bovine Serum (Hyclone, Thermo Fisher, www.hyclone.com), 10% v/v CMG supernatant (the source of CSF-1), 2 mM glutamine (UCSF Cell Culture Facility), 110 µg/mL sodium pyruvate (UCSF Cell Culture Facility), penicillin and streptomycin (UCSF Cell Culture Facility). The bone marrow cells were cultured for 6 days at 37°C with 5% CO2. The cells were harvested with cold PBS (without Mg and Ca), and frozen in BMM+10% DMSO.
For infections, H. capsulatum cells were grown to late log phase in HMM. The cells were pelleted and resuspended in BMM medium, sonicated twice for 3 seconds, and counted by hemacytometer. Approximately 1×106 H. capsulatum cells were used to infect 2×105 BMDM in 24-well cell culture dishes. After a 1 hr incubation, the infected macrophages were washed twice with D-MEM High Glucose and then incubated in 500 µL fresh BMM medium with or without 100 µM FeSO4 at the zero time point. At 6, 24 and 48 hrs, the medium was removed from the wells and 500 µl H2O was added. After 5 min incubation, the lysed macrophages were transferred to a 1.5 mL eppendorf and sonicated for 3 sec. Dilutions were plated onto HMM plates to determine CFUs. CFUs were normalized to the zero time point. Significance was determined using ANOVA with Bonferroni Multiple Comparisons Test.
Competitive index mouse infections
H. capsulatum strains hcLH95 (wild-type), hcLH97 (sid1Δ::hph), and hcLH106 (sid1Δ::hph+SID1) were grown in 5 mL of HMM. Cells were passaged once and grown until late log phase. 1 mL of culture was sonicated twice for 3 seconds to fully disperse cells then washed twice with cold PBS. Cell concentration was determined by hemacytometer. Mice were infected intranasally with 5×104 cells in 25 µL PBS containing an equal proportion of hcLH95 and hcLH97 (wild-type and sid1Δ::hph) or hcLH95 and hcLH106 (wild-type and sid1Δ::hph+SID1) followed by 5 µL of PBS chase. Four mice were used for each time point.
Lungs were homogenized in 5 mL of HMM. Serial dilutions were plated onto both HMM and HMM+hygromycin plates in order to distinguish sid1Δ::hph and sid1Δ::hph+SID1 strains from the wild-type strain. Enumeration of wild-type, sid1Δ, and sid1Δ+SID1 yeast allowed for the determination of competitive index ratio (CI) using the following formula: CI = (mutant output/wild-type output)/(mutant input/wild-type input). Input was determined at four hours post infection. Significance of the results was determined with the Kruskal-Wallis Test (ANOVA) with Dunn's Multiple Comparisons Test and with the Mann-Whitney/Wilcoxon rank sum test. Enumeration of yeast by patch test onto HMM and HMM+hygromycin was also performed and yielded a CI identical to the plating of serial dilutions (data not shown).
Female C57BL/6J mice were purchased from The Jackson Laboratories. Experiments were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at U. C. San Francisco.
Supporting Information
Table S1.
Primers Used in This Study
Figure S1.
Iron Regulated Gene Expression of Siderophore Biosynthesis Genes in G186AR. Quantitative RT-PCR of siderophore biosynthesis genes in G186AR (hcLH95). Total RNA from cells grown in the presence or absence of 5 µM FeSO4 for 1, 4, and 24 hrs was analyzed for relative transcript levels using QRT-PCR. Transcript levels were normalized to ACT1 transcript. Relative quantities were normalized to t = 1 hr+Fe values. For the 1, 4, and 24 hr time points, white bars indicate growth in medium supplemented with FeSO4 (+Fe), and gray bars indicate growth in the absence of FeSO4 (no iron).
We thank Erica Dirks for help with plasmid construction and transformations, Mark Voorhies for invaluable assistance with sequence analysis and statistics, Davina Hocking Murray for assistance with figures, and Diane Inglis for advice and technical assistance with mouse infections. We are grateful to William Goldman and Bruce Klein for their generous gifts of strains and plasmids. We thank Michael Shiloh for advice regarding statistical analyses. We are grateful to Jeffery Cox, Hiten Madhani, M. Paige Nittler, and members of the Sil laboratory for helpful comments on the manuscript and for useful discussions.
Author Contributions
Conceived and designed the experiments: LH JM JR AS. Performed the experiments: LH JM. Analyzed the data: LH JM. Wrote the paper: LH AS. Provided editorial comments: JM and JR.
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text:
Mission Statement
The mission of BRT Extrusions Inc. is to:
- Provide a clean, safe and injury free work place for all of our employees and vendors.
- Offer a friendly environment where all employees are treated with respect and dignity, and are valued team members.
- Manufacture a quality product for our customers and deliver our product on time.
- Address issues consistently, fairly and in a timely manner and react to all questions and concerns of our valued customers and employees.
- Grow our business allowing us to achieve our financial objectives and provide employment security for all team members.
- Maintain each and every customer.
Do it right the first time and repeat it each and every time thereafter.

subset: commoncrawl


text:
by Shannon Lush
Throughout its 50 years, 'Doctor Who' has mined hundreds, perhaps thousands, of pre-existing songs, films, books, oral and written traditions, and established world history for source material. From the 'historical', stories of the First Doctor's era that would see the TARDIS crew encounter well-known figures from history such as Emperor Nero and King Richard the Lionheart, to the Jason and the Argonauts-parallels to the Fourth Doctor story 'Underworld', the adventures of The Doctor have in some way or other been inspired by outside elements.
Due to its nature as a British series, it is therefore natural that 'Doctor Who', chiefly the product of British writers, actors, directors, and producers, has explored subject matter to which these folks consider important and reflective of British history and culture. Script editor Robert Holmes peppered his Season 15 story 'The Sun Makers' with numerous satirical references to the British tax system, for example. British history spans thousands of years, but what subject could be more alluring to 20th century Whovians than that of the world-recognized, tragic tale of the British liner 'Titanic'? Of all possible topics for 'Doctor Who' to tackle, this would seem to be a natural fit; the subject matter is so well known to modern audiences that many can recount 'some' detail that has been embedded in pop culture by this point. The ship's port of call, her intended final destination in New York City, her Captain's name, the relevant date of April 15th, 1912...
Yet, for the most part and with rare exception, the 'Titanic' has rarely played a role within the televised 'Doctor Who' universe. Most notably, a space ship named after her featured prominently in the Tenth Doctor's era, during the events of the Christmas Special entitled 'Voyage Of The Damned'. The plot in its basic form featured the Doctor preventing this namesake ship from crashing and suffering the same fate, symbolically, as the original. In 50 years of 'Doctor Who' stories broadcasted on television, this is the only time that the 'Titanic' played a major role, and even then it was not truly the ship herself, merely a futuristic spaceship with the same name. It is perhaps testament to the residual scarring of the national British psyche that occurred after the original tragedy that, during post-production of this story, producers were obliged to 'spoil' the ending by revealing that this 'Titanic' does not 'sink'.
However, the 'Doctor Who' connection to 'Titanic' did not begin there. Earlier, the Fourth Doctor offered a downbeat, and somewhat morbid, comment during the events of the story 'Robot'. He stated he didn't care for the word 'unsinkable', and when his companion Harry Sullivan questioned this belief, The Doctor reinforced it by remarking 'Said the iceberg to the Titanic, gloop, gloop, gloop, gloop'. Much later still, she is invoked to demonstrate the shadowy nature of The Doctor. The character Clive in the story 'Rose', the debut episode of the 'New Series' begun in 2005, shows Rose a photograph of the Ninth Doctor standing before 'Titanic' herself prior to her maiden voyage. Clive remarks that as the story goes, The Doctor had convinced a family not to sail on her. The implication is The Doctor therefore saved their lives as they escaped the fate that nobody at the time was aware would befall the ship..nobody, of course, except the time-travelling Doctor.
Other than these incidents and in-story mentions, 'Titanic' did not play a part in televised 'Doctor Who', perhaps chiefly due to the sensitivity involved in locating a story aboard a ship that must, by worldwide historical knowledge, sink. The Doctor saves lives, after all, and in this situation, he simply cannot, otherwise the bounds of credibility that allow such a series to skirt the edges of known historical records would collapse. The preferred term used on-screen to limit the Doctor's involvement in affecting lasting and meaningful change in historical events is 'time locked'; in the case of 'Titanic', given how it is such a well-known part of British history yet has not been afforded even one story set partially or fully aboard her in 50 years of 'Doctor Who', the term seems to encompass the writing and production teams of the past, as well. Whether or not any have ever considered a story featuring 'Titanic', none have been willing to produce such a story.
This is, however, not the end of the 'Titanic' connection to 'Doctor Who'. Despite the lack of on-screen adventures, there are notable examples of both Expanded Whoniverse work as well as material featuring 'Doctor Who' performers who also have worked on material documenting 'Titanic'. While this is not an exhaustive list, (as Expanded Universe material is continuously being produced and therefore future releases may indeed delve into the subject matter more thoroughly), some of the more notable material includes, perhaps, among the best.
'A Night To Remember', the 1958 British film adaptation of the 1955 nonfiction bestselling book of the same name by Walter Lord, is widely considered, like the book it is based upon, as the definitive account of the 'Titanic' tragedy in its medium. Walter Lord himself as a young child in England had witnessed 'Titanic' leaving port. The awe it filled him with would years later inspire him to seek out survivors and produce a fact-filled book of high journalistic integrity and investigative material. The book stands today as the definitive account in the eyes of 'Titanic' historians, of both the armchair as well as professional variety. The 'Doctor Who' connections to the several media adaptations of Lord's book are numerous.
The film was primarily shot in Pinegrove Studios, slightly over five years before those very studios would become home to 'Doctor Who'. It features a large number of actors who would go on to play memorable characters in 'Doctor Who'. Billed as the film's co-star was Ronald Allen, who appeared first as Rago in the Second Doctor's story 'The
Dominators', then as Professor Cornish in the Third Doctor's 'The Ambassadors Of Death'. Honor Blackman would go on to match wits with the Sixth Doctor as Professor Lasky in 'Terror Of The Vervoids', part of the 'Trial Of A Timelord' that comprised the entirety of Season 23. The afore-mentioned 'The Sunmakers' is represented, with the actor Richard Leech appearing in this film as well as there, as the character Hade. Ralph Michael would later appear as Balaton in another Fourth Doctor story, 'The Pirate Planet', written by script editor Douglas Adams.
Harold Goldblatt portrayed Professor Dale in the Third Doctor story 'Frontier In Space', a store widely considered a 'prequel' to the story that followed, 'Planet Of The Daleks'. Philip Ray played Eldred in 'The Seeds Of Death', from the Second Doctor's era. An uncreditted young actor by the name of Jeremy Bulloch, decades before the role of Boba Fett in 'Star Wars' would transform his life, also appears briefly. He would go on to feature in two 'Doctor Who' stories, 'The Space Museum' as Tor during the First Doctor's era, and 'The Time Warrior' as Hal during the Third Doctor's time.
The greatest connection between the film and the production universe of 'Doctor Who', however, is the appearance of two actors who performed extremely admirably in the former and boast quite interesting accolades in the latter. Jack Watling, a well-regarded actor often in demand in the British film and television industry of the 1950's and 1960's, played Professor Travers in the Second Doctor classic story 'The Abominable Snowmen', as well as its sequel-of-sorts, 'The Web Of Fear'. The latter story has just recently been re-discovered after a near-fifty year absence as one of the 'lost' stories, and was reviewed in depth in the companion podcast series to this blog. Its special status as a lost story regained and re-released during the 50th Anniversary celebrations of 'Doctor Who' was instantly assured as it climbed the iTunes charts within minutes of being made available for purchase on that platform. Jack Watling, in addition to his distinguished acting career, also of course was the father of the actress Deborah Watling, who portrayed the companion character Victoria Waterfield during the Second Doctor's era, and twice during that period acted alongside her father in these stories featuring 'The Great Intelligence'.
Geoffrey Balydon, who appeared in both 'A Night To Remember' as well as the Fourth Doctor story 'The Creature From The Pit' as the character Organan, remains notable simply for the unique 'alternate Doctor' factors that dominate his career. In addition to being seriously considered for the role of the Fourth Doctor prior to the casting of Tom Baker, he went on to play an alternate Doctor in the 'Doctor Who Unbound' series of audio plays from Big Finish Productions, the central theme of which concerned untold tales drawn from a myriad of possible or potential outcomes to established 'Doctor Who' canon. In Balydon's stories he portrayed not an alternate Fourth Doctor, but instead a very different First Doctor.
The 'Night To Remember/Doctor Who' connections do not end with the film version of the story, however. As with most creative products, the book spawned not merely a film comprised of numerous future 'Doctor Who' actors, but an audio book as well. In this instance, the abridged story was narrated by an actor familiar to Whovians for several guest star appearances. Martin Jarvis began his association with 'Doctor Who' as the character Hilio in 'The Web Planet' during the second season of the series, and returned to play Butler in the dreadful 'Invasion Of The Dinosaurs' during Jon Pertwee's tenure. It is as the Governor, however, in the Sixth Doctor's era in 'Vengeance On Varos', that many longtime fans will recall him, due to that character being, arguably, the hero of the piece. Bound by duty, conflicted by feelings of mercy and ultimately redeemed by the climax of the tale, Jarvis brought the Governor to life in a highly-memorable manner, and that story ranks among the best of Colin Baker's all-too-short era.
Jarvis brings authoritarian tones, dignified bearing, and ability to embody the various real-life people he is called upon to mimic to the audiobook version. His work stands as an excellent companion piece both to the book upon which it is based as well as the film. Clearly, the reluctance to tackle the subject matter of such a tragic event in British history on-screen only extends that far; behind the scenes, the long list of actors who transitioned from portraying ill-fated passengers and crew of 'Titanic' to nomads, military officers, professors, futuristic villains and monsters in 'Doctor Who' cements the connection to two iconic British topics.
To this point, 'Titanic' has featured prominently in the Expanded Whoniverse on only one occasion, the 'New Adventures' novel 'The Left-Handed Hummingbird', by Kate Orman. It remains the only example of the fictionalizing of the world-known event, and the only novel to locate the majority of events aboard the great ship. And so, 'Titanic' and 'Doctor Who' remain linked in these ways, thanks to the excellent work of the many creative people who graced both topics with their luminous work.


text:
To live the life you want, take an active role in your sleep therapy.
Sleep apnea therapy can help you sleep better at night and be more active during the day. The free DreamMapper* app and web-based software can help keep you motivated.
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text:
Snow is predicted for the next two days.
At the beginning of Winter
I decided to embrace the cold and the snow.
That lasted until a couple of weeks ago.
Now I have to say.....
I've had enough!
From each and every window the view is the same...
Oh my, this opens up a whole new world of digital creativity for me.....imagining the possibilites! Stay tuned for more.


text:
Philadelphians know what Wawa does best: shorties; coffee; iced tea; scrapple and, of course, surcharge-free ATMs.
Almost 15 years after Wawa plopped its first no-fee ATM into one of its stores they will celebrate one billion surcharge-free transactions Wednesday.
In traditional Philly style, the fine people over at Wawa are teaming up with banking partner PNC to mark the moment with a Mummers parade and party. The "Billion Dollar Savings Parade" will start at Love Park in Center City.
"While most people associate Wawa with quality products and the coffee and hoagies they can't live without, for many customers, Wawa's surcharge-free ATMs are a way of life," said Wawa President & Ceo Howard Stoeckel.
Wawa claims that waiving ATM fees in their stores has saved customers about $1.3 billion since they started the practice in 1996.
"We have three times the traffic to ATMs in our stores than all other banks in the region have to their ATMs," Stoeckel said. "Our ATMs have truly become a source of convenience and savings that our customers can't live without and we are thrilled to celebrate our commitment to providing customers with the highest level of value and convenience possible."
The party starts at Love Park at 10:30 a.m. The Mummers, along with Philadelphia Police, local mascots, dignitaries and Wawa employees, customers and the Wawa mascot Wally the Goose will march their way up to the Wawa at 17th and Arch Street so that Franklin can get his money without the fee.
And some lucky customers will even get something for free if they don't make it to the event Wednesday. Wawa ATMs at all 570 stores will randomly be spitting out "golden coupons" for a free Wawa iced tea or fruit drink.


text:
Tin can 175ml metal.
Tin can 175ml metal.
Salesprice with discount
Sales price € 1,49
Sales price without tax € 1,22
Tax amount € 0,27
Content: 175ml
Length: 75 mm
Width: 88 mm
Content: 175ml
Length: 75 mm
Width: 88 mm
Height: 39 mm
These tin cans of 175ml are available in different designs.
The cans are sold with a plastic cap which can be placed after the can is filled. Your liquids are air sealed packed this way.
One side is unprinted. This way You can use Your own label.
Its possible to buy the cans filled with EVOO. Just select Yes or No when You order these cans.
There are yet no reviews for this product.


text: BRIDGEPORT—Catholic schools students throughout the greater Bridgeport area participated in last Friday's St. Patrick's Day Parade in downtown Bridgeport. The students proudly paraded their school spirit as they marched through downtown Bridgeport. Hundreds turned out for the parade. The day for many area Catholics also began with Mass at St. Augustine Cathedral.


text:
I am about to write a 3rd party module which should add/remove users to the allow {} block in the config.
The reason is, that I want a highly restrictive ircd where the admin user can add/delete users from his irc-client e.g. by using /adduser
Can someone help me if this is possible to change values (e.g. allow) in the configuration (unrealircd.conf) or has another idea how to implement this?
Changing config settings within client
Need a module converted from 3.2.x to UnrealIRCd 4? Or looking for a certain module? Ask here.
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text:
Mass media 10
What's in this video?
Social media presence is a critical part of any business. It elaborates the role of social media in the modern business world. This video encourages viewers to believe in the today's technological innovations that surrounds the social media environment. It provides you a better understanding of how online business and social networking has evolved and how businesses can use internet to make connections and gain economic leverage.
Creative Concept
By selecting the appropriate tools, this medium can easily be disseminated to the right audience. Great advertising strategies ensure that all media communications maximizes its effectivity. It means that it is s not only about the destination of the message but also to reach them with best result.
Having a video presence is crucial in today's marketing mix, with so much presence now being online. Using an Advertising Video Production is such a great way to capture that market when promoting your business online. Business video productions needn't cost your business an arm and a leg. Instead TuberADS provides you a video ad production well within your means, beckoning you to come back more regularly to keep your advertising fresh for your audience.
Suited for:
This will work for social media managers, online marketers, PR practitioners, advertisers, companies that are involved in internet activities.


text:
Designed by German company Schuermann Architects, the velodrome's outstanding feature is its spectacular dome-shaped roof.
The venue seats 6,000 and will host all Olympic track cycling events on its 250m purpose-built oval. The mountain bike and BMX events will take place nearby.
Work began on the venue on 30 October 2004 and is on course to be finished by early 2008.
What are these?


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Paragould, Arkansas resident Nathan Musgraves, an employee of Union Pacific, filed a Federal Employers' Liability Act suit in the Circuit Court of St. Clair County, Illinois alleging he sustained personal injuries after being hit in the back by a door.
Musgraves says he was working as a tie plug operator for Union Pacific's Maintenance of Way Engineering Department and had transferred to the railroad's facility in Dupo, Illinois when the incident took place on December 23 of last year.
According to the complaint filed on July 23, Musgraves' duties involved him entering the yard, shop, or facility and use an 18 wheeler box trailer to retrieve a switch heater. While he was entering the box, the wind caught the door and slammed into his back.
Musgraves alleges that he sustained permanent disfigurement and disability due to his injuries. He also alleges that a pre-existing condition was aggravated by the incident and required surgical treatment.
The suit alleges negligence on the part of the railroad for its failure to provide a latch to secure the trailer door. Musgraves seeks damages in excess of $100,000.


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Dr. Shelly Tochluk
Monday, July 11, 2016, 7:00 P.M. Faith United Church of Christ will host Shelly Tochluk, PhD., Chair, Education Department, Mount Saint Mary's University, Los Angeles, California. She will be sharing information from her latest book, "Living in The Tension: The Quest for A Spiritualized Racial Justice"
An educator, with a background in psychology, Dr. Tochluk spent ten years as a researcher, counselor, and teacher in California's public schools. She now trains teachers to work with Los Angeles' diverse school population as Chair of the Education Department at Mount Saint Mary's University. Her personal dedication to confront issues of race developed first through her participation with UCLA's NCAA Division-1 All-American Track and Field 4X400 meter relay team and later through her inner city teaching experiences. She currently works with AWARE-LA (Alliance of White Anti-Racists Everywhere-Los Angeles). With this group, she co-created a workshop series that leads white people into a deeper understanding of their personal relationship to race, white privilege, and systemic racism.
A limited number of Living in the Tension: A Quest for a Spiritualized Racial Justice may be purchased at the church office for $20.00. Please call 319-338-5238 or email firstname.lastname@example.com to arrange for the pick-up of a book.
Vivian Hahn, a talented Iowa City artist and Faith UCC member, has been drawing for many decades. She will be holding her first community art exhibit at Faith United Church of Christ. Vivian's artwork will be on display at Faith UCC throughout the month of June.


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Ideally, you'd be able to empty the coffers for dad on Father's Day. If money was of no object, you could rent out an amusement park or buy him a little quaint island he could retire on. But, if you're working with a budget, you can still get and give him roughly the same feeling any of these thoughtful gifts under $50.
To help you with your shopping, we've put together 40 gifts that fit the bill. Even if you just grab a comfortable throw blanket he can keep and head over for a movie and popcorn, dad might just love the gesture most.
Below are 40 gifts under $50 your dad will love for Father's Day:
An Omaha Steaks gift box
Whether dad likes grilling steaks or just loves eating them, a sampler of delicious meat is likely to be one of his favorite gifts to date.
Omaha Steak Entertainment Sampler, $49.99
A Hydro Flask water bottle
Perfect for everything from camping to everyday use, this Hydro Flask water bottle is the real deal. It's our pick for the best water bottle you can buy, and during Outdoor Gear Lab's testing, ice resisted melting for nearly a full 24 hours, and water was ice cold for more than a full day.
Hydro Flask 32oz. Wide Mouth, $39.95
A Harry's shaving kit
If your dad could stand to take more time for himself, setting him up with nice tools for a quick but indulgent routine is a great gift. Especially with the option to engrave (and especially if dad is still using disposables), Harry's Winston Shave Set is a thoughtful and practical gift you can feel good giving.
Harry's Winston Shave Set, $25 (+15 to engrave)
A waterproof speaker
Perfect for the summer months spent at the beach, barbecuing by the pool, or simply for the demands of the rugged lifestyle, a waterproof portable Bluetooth speaker is a great gift for dad.
JBL Clip 2 Waterproof Portable Bluetooth Speaker, from $39.90
The Echo Dot
Alexa can tell dad jokes or the forecast, order him pizza, and play him music, among lots of other helpful skills.
Amazon Echo Dot, $39.99
A card that shows you appreciate his dad humor
No matter what gift you decide to give dad, he's probably going to be most thankful for the thought you put into a day that's just about celebrating him. If you want to make his day (and give him something he can hold onto if he wants), a fun card is a great addition.
Dad Jokes Card, $3.88
A tablet he'll love using
For casual internet perusing, Facebook updating, and game playing, Amazon's Fire tablet is a tough value to beat. It isn't exactly high tech, but it's $40 and it works. If dad isn't a self-proclaimed tech fiend, it'll do what he needs it to do.
Fire 7 Tablet with Alexa, 7" Display, 8 GB, $39.99
A nice leather case for his phone
An understated, rugged leather case will keep his phone safe and hopefully blend in seamlessly with the rest of his gear. He might not have many accessories, so why not pay extra mind to the ones he does own?
Nomad Rugged Case, $44.95
A make-your-own hot sauce kit
Especially if your old man is big into the make-your-own movement, he'll probably have a lot of fun mixing his own hot sauces — whether they turn out well or not.
Make Your Own Hot Sauce Kit, $34.95
A beautiful stainless-steel French press
If dad loves coffee, he probably won't turn away a beautiful stainless-steel French press from Williams Sonoma on Father's Day. If coffee is a hobby, luxury, or necessity, it has something to offer.
Williams Sonoma Stainless-Steel French Press, $49.95
An external battery
Perfect for dads accustomed to running around, a portable charger will keep his gadgets ready to go whenever he needs them.
Jackery Bolt 6000 mAh Portable Charger,$29.99
A fancy shaving cream
Whether dad steals your mom's nice lotion or might like trying something new, grabbing a nice shaving cream that smells like sandalwood is an easy way to get dad into some more daily luxuries.
The Art of Shaving Sandalwood Shaving Cream, 5 oz, $25
A stainless steel tumbler to keep his hot drinks hot and cold drinks cold for hours
This stainless-steel vacuum-insulated tumbler was built to withstand the extreme conditions of the outdoors, and the overpreparation for day-to-day life is wonderful. This will keep your dad's cold drinks cold and hot drinks hot for hours on end.
YETI Rambler 20 oz Stainless Steel Vacuum Insulated Tumbler, $29.99
A Tommy John gift card for comfortable clothes he can pick himself
Tommy John makes some of our favorite comfortable loungewear (as well as basics like underwear, which is where most of their recognition comes from). You might not know exactly what to get your dad or in what size, but a gift card lets him look around for himself (with you still covering the tab).
Tommy John Gift Card, $50
A breakfast sandwich maker
If he loves breakfast sandwiches, he's probably going to be pretty enthusiastic when he opens this. It might seem gimmicky, but it has over 2,000 reviews on Amazon and a 4.6 rating.
Hamilton Beach Dual Breakfast Sandwich Maker, $34.15
A Neil deGrasse Tyson book
If dad loves reading, science, or Neil deGrasse Tyson, this is sure to be a great pick.
Astrophysics for People in a Hurry Hardcover by Neil deGrasse Tyson, $11.37
Bourbon-infused coffee
If he likes bourbon and/or coffee, this is a thoughtful, unique gift he's not probably going to have bought himself or found on his own.
Bourbon Infused Coffee, $20
His new favorite summer shoes
Do your dad a big favor and pick up what will undoubtedly become the pair of shoes he spends the whole summer in. Comfortable, breathable, lightweight, and travel-friendly, they'll go anywhere he goes.
Soludos Men's Original Classic Stripe, $48
A leather passport holder
For dad's big upcoming adventures, a leather passport cover and luggage tag (which you can engrave) is likely not something he'd pick up for himself but will love having.
Leatherology Passport Cover + Luggage Tag, $50 (+$20 for engraving)
A gift set of fun socks
Socks are every outfit's perfect opportunity for a guy to show some flair. If dad might appreciate some cool socks from a new brand, you might want to check out Happy Socks' Father's Day Gift Box.
Father's Day Gift Box, $36
A beer-making kit
If your dad might get a kick out of making his own beer (or, better yet, making it with you), then this a fun experiential gift he'll definitely remember for years to come.
Mr. Beer Premium Gold Edition Homebrewing Craft Beer Making Kit, $48
A Himalayan salt block to spice up his cooking
Does your dad like to cook (or does he just like to eat good food)? Salt blocks like this one give meals an enhanced flavor that can't be mimicked by a frying pan. Plus, the minerals in Himalayan salt are supposed to give a more nuanced flavor than regular table salt, and the amount of saltiness will be regulated by the type of food (moist food absorbs more, fatty foods repel it).
Himalayan Salt BBQ Plank, $29.99
"Game of Thrones" Monopoly
Use this to give a nod to his "Game of Thrones" obsession and institute a family game night.
Monopoly: Game of Thrones Collector's Edition Board Game, $38
Really nice face cream
If he isn't married to a skin care routine yet, he might love Kiehl's — it's nicer than what he'd pick up at the drugstore, but not so expensive he can't bring himself to replace it when this pot runs out.
Kiehl's Ultra Facial Cream, from $29.50
A craft beer flight holder
The man cave most likely doesn't have this yet, but it's perfect for entertaining or simply getting more enjoyment out of the hobbies and drinks dad loves.
Craft Beer Flight Holder, $49.95
A body wash he can't find at the drugstore
Nicer than what he might pick up at the drugstore, this MALIN+GOETZ body wash smells great and uses natural glycerin and amino acid technology to cleanse and hydrate.
MALIN+GOETZ Rum Body Wash, $36
A charging dock for his phone and Apple watch
A simple, all-in-one charging dock for his essentials that won't clutter up his space.
Griffin WatchStand Charging Dock for Apple Watch and iPhone, $23
A tobacco- and amber-scented candle
This tobacco- and amber-scented candle will make a nice addition to his desk or man cave.
We Took To The Woods Whiskey River Candle, $37.98
A dopp kit
A nice dopp kit goes a long way in making Dad's life easier and his essentials more organized. Dagne Dover's neoprene makes this look much more luxe than its price suggests.
Dagne Dover Small Hunter Neoprene Toiletry Bag, $35
An engraved money clip
Understated, elegant, and personalized, this money clip looks nicer than something you ordered for less than $20.
Personalized Money Clip, from $18
A mug that expresses just how much you love him
Whether you're indulging in your old man's love for Star Wars or just getting the point across in a lighthearted way, he'll love seeing this in his cabinet and thinking of you for years to come.
Worlds Greatest Dad Star Wars Mug, from $15
A drone
For the man who has everything.
National Geographic Quadcopter Drone, $39.99
A gift card to Patagonia
If dad loves the outdoors but might not need another of whatever you select for him from Patagonia, you might put the purchasing power in his hands for a change. You don't need to haunt your mother with requests for taking inventory of his working or not-working gear, and he can shop for the latest edition of something or replace the old.
Patagonia Gift Card, $50
A beard oil
If Dad is letting his stubble grow out, or he's already sporting a beard, this is a grooming product he should have, but might not.
Jack Black Beard Oil, $25
A media streamer
If you want to gift him the joy of streaming, this is what we'd suggest picking up. You can read our review for the full rundown, but in summary, this is nearly as fast as competitors twice as expensive, it supports as many apps as you'll find on a streamer, and its interface is super straightforward.
Roku Express HD Streaming Player, $29
A Blue Bottle Coffee subscription
Grab dad a high-quality blend he might not have tried but will likely really enjoy. Blue Bottle Coffee focuses on honoring flavor profiles, finding the best beans in the world, and shipping those beans to you within two days of roasting so they always taste their best. You can read our full review here.
Blue Blue Coffee Voyager Pack, $17.50
A personalized coupon book he can redeem
This personalized Father's Day coupon book is sweet and thoughtful, but more polished than if it was entirely homemade. You can gift him his choice of TV, free take-out-the-trash days, or anything else that might make his next few months better.
Personalized Father's Day Coupon Book, $9.98
A backpack cooler
This cooler is perfect for when a picnic or tailgate party involves more gear than he can carry in two hands. Its 20-liter capacity will hold a 12-pack plus ice, and two side pockets with adjustable toggles can handle wine or water bottles.
Eddie Bauer Bygone Backpack Cooler, $35
A good pair of earbuds
You don't always have to pay an arm and a leg to get something great in the tech world. We're big fans of these inexpensive Xiamoi earphones, and we think he will be too. Whether it's for music or audiobooks while he works out or mows the lawn, your dad is sure to be grateful for some great new earbuds.
Xiamoi Hybrid Dual Mi Earphone, $25.99
A cookbook that is one long "ode to the Southern breakfast"
This is basically the how-to book for every mouth-watering Southern breakfast dad might ever want, and he'll probably have a lot of fun making his way through each one.
Big Bad Breakfast: The Most Important Book of the Day, $20.40
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February passed quickly.
Maybe too quickly. But, also maybe not.
I now live alone.
Except for the cat.
I have never lived alone before. I've never been the sole owner of a space bigger than just one room. I have never been responsible for so many payments and bills and just adult stuff as I am right now.
And although I've done it and it's all set up, running smoothly and I can do it; I actually feel really fucking overwhelmed.
I look back on my posts from last year, the pictures, the comments, and I cannot really see anything to indicate that at this point, a year on, I would be completely changed.
And I mean: COMPLETELY.
I don't even think it's just my life that has changed, it is ME.
I feel pumped like I've just run a marathon and I'm high on adrenaline. Like I'm ready to take on anything and win. But standing back and looking at myself and reflecting on that shit, just blags me. Totally blags me.
Who am I? When did I become this person?
I keep saying this: I must start blogging regularly again. I must! And then a whole month passes and I really feel as though I just blinked and missed it. I really do.
I have a massive wishlist of things to do. And some of those things are already underway, but it's a massive waiting game at the minute too. As soon as I can share, I will.
For now, just keep waiting.
Find me here too!


text:
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DescriptionDelicately dainty, these exquisite 10kt white gold earrings feature a sparkling blue topaz surrounded by shimmering diamonds. perfect for an evening out on the town.
Product Details
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Customer Reviews
2 Reviews
06 Aug 2017 Magp1G1rl Melbourne
Nice earrigs
Had to replace them after claiming in insurance as one fell out of ybear. Very disappointing considering the price. Asked in store about extra reinforcement and was told they can only tighten the stoppers with their pliers. I can do that myself.
26 Nov 2015 Benjamin Sydney
Colour not what was expected
Not happy that the colour in the picture is not the same in real life, In real life the blue is extremly light in colour.
Find In Store


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Today my brain is very far away, dreaming of going back to Italy.
All photos by Giacomo Pulcinelli.
What are you guys up to this weekend? My plans include making more candy pins, seeing Harry Potter (finally!), possibly a visit to the hair salon, and unpacking the last boxes.
Friday, August 19, 2011
Happy Friday
4 comments:
These shots are amazing! The first is absolutely breathtaking.... my weekend will be full of relaxation and baking. Hope you have a good one. :) Sea MarieReplyDelete
After seeing these awe-inspiring photos I simply forgot what I was up to for the weekend!ReplyDelete
Whoa! Crazy. Those hills are so ripply they look like some great, green blanket!ReplyDelete
Your post makes me want to get online and book some plane tickets. Beautiful photos!ReplyDelete
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After Vince Cable's embarrassing claims about EU leaders, have the Liberal Democrats had it?
John Longworth, co-chairman of Leave means Leave and former director general of the British Chambers of Commerce, says YES.
There have been some pretty big lies and exaggerations surrounding Brexit.
We were told that the referendum would increase unemployment by half a million, that there would be a recession, that Brexit would be bad for the regions. None of these have come to pass. Many of these and other claims were made by the Cameron government and the Remain campaign. However, when it comes to being economical with the truth, the Liberal Democrats take some beating.
Vince Cable, whom I respected, is now even scraping the barrel with false claims that foreign leaders support a second referendum.
The Lib Dems are stooping so low as to make common cause with foreign powers to undermine the democratic will of the British people and the government in its negotiations, thus making Britain worse off. Gladstone, the great advocate of liberal markets and free trade, is reeling in his grave.
The Lib Dems may not be finished as a party, but their credentials as liberal or democratic are certainly at an end.


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In video games short history as a medium, much time has been spent both disparaging them for their limitations and exonerating them for their potential. As someone who has grown weary of the many debates about games, I can honestly say I get it. I get why some people think videogames are ridiculous. For every copy Journey or Minecraft it seems like there are a hundred more bloody bikini clad torsos and well-dressed men murdering sexy nuns. In response to N+1's challenge to game's artistic merits, Tom Bissell wrote, "The games I am most interested in allow me a way out of myself …. [they] allow me to observe and control fictional characters, and when I am at the helm, I try to make these characters behave not as how I would, but how I feel they would want to—a strange sympathetic process for which there is, as of now, no good name." Leaving alone the tired "are games art" debate Bissell highlights one of the most valuable services that games can provide: they can give us a window into someone else's world. They can teach us empathy.
There seems to be a growing trend of late for games to explicitly attempt to encourage players to empathize with life situations of others. Papo & Yo, despite all its imperfections, serves as a harrowing look into the world of a boy living with an abusive alcoholic father. The Binding of Isaac explores the world of a boy enduring religious abuse. Dear Esther let's us explore the memories of a man who lost his wife, Amnesia: The Dark Descent gives us a view to another's dark past and latent fears, and To the Moon gives us a glimpse into the romantic difficulties of people with Asperger's syndrome.
And while such experiences illustrate the humane side of our misogynistic and narrow-minded medium, it is important to recognize how limited these glimpses into the lives of others are. We can envision a boy running from an enraged father when Monster eats a frog, but we are not walking in Vander Caballero's shoes nor do we want to. As we solve puzzles and flee from otherworldly terrors, we are ever aware that we are invested in fantasy. And while these fantasies might correlate to a person's real life experience, for those of us who are privileged, there remains a gap in our empathy: we maintain enough cultural distance from such things to assume they are rare. Educational Simulations' Real Lives 2010 seeks to bridge this gap by giving us a view into the lives of others based entirely on statistics.
Players begin the game being randomly born into a family chosen from census data of 2010. Initially all you can do is "age a year," but as the game progresses, you are given more and ways to shape your character's future and make decisions for her or him. You can move out of your parents house, attempt to go to college or trade school, get a job, search for a romantic partner, borrow or invest money, try to have a child, start a business, change houses, adjust monthly spending, and even immigrate to another country. Your successes and failures in these endeavors are determined both by your character's ratings on traits like strength, intelligence, and happiness, as well as your family, cultural, and financial situation. And each time you age a year, you will be informed of various major events in the life of your family, things like sickness, raises, pregnancy, and marriage proposals. Some of these events are merely informative, while others give you options. For instance if you become pregnant, you can have the child, give it up for adoption, or have an abortion. Some of Real Lives most important moments, however, come not from player choices but what happens to the player's character when she ages a year.
My characters, have suffered spousal abuse, been drafted into the military, struggled with alcohol abuse, suffered depression, lost parents and children to cancer, come down with diabetes, lost and gained money in the stock market, immigrated to first world countries, had children, received pay cuts and raises, enrolled in and been pulled out of school by parents, and been raped. I have played as an artist in Burkina Faso, a devout muslim in Lybia, and shoe maker in China.
Given that Real Lives functions a simulation and a text adventure, the players "experiences" may fall upon them rather coldly. I am told that my character has been raped but not really given any indication as to how that life experience has affected her. I see that her "happiness" stat has fallen from 47 to 16 but that seems a crude way of expressing her experience. I feel powerless to help her. I can only make decisions about how she will spend her time and money and if and when she might marry. I cannot control how this tragedy will shape her.
When the words, "you've been raped" appear I just stare unblinking at the screen. There is no window into the thoughts and emotions of an impoverished artist in Burkina Faso whose already fragile life is subjected to the most degrading and debilitating of assaults. This kind of pisses me off, so I turn off my computer and go to bed.
When I wake up, I begin to think about what a game that thoughtfully explored the effects of rape would look like. I think about the controversy surrounding the upcoming Tomb Raider game and how that game surely is not the right venue to tackle an issue that is so painful for so many. As my anger at Real Lives 2010 subsides, I realize that while it may not explore the emotional toll of rape, it manages to do something more immediately helpful. It had forced me to acknowledge the life experiences of an impoverished artist in Burkina Faso. I don't like to think about things like rape and abuse, but I know that Real Lives' systems are based entirely on real world statistics and probability. I must acknowledge that my character had these experiences–experiences that I would not wish on my worst enemy. And she had them not because I failed as a player but because the odds were stacked against her.
I like to think I am aware of how privileged I am, but Real Lives forces me to acknowledge that this could not be further from the truth. As I have played, I have been surprised by how difficult it has been to help my characters realize their dreams. My first character was very artistic and I wanted to send her to art school. Her parents pulled her out midway through primary school to help around the house. I managed to land her a job as an artist for a time but had quit in favor of a higher paying job so as to afford to feed her two children. I had hoped to help my Libyan character start his own business but could never quite afford to invest in the company consistently and health problems due to diabetes made him a hindered his ability to manage his business.
In real life, I live a fairly normal lower middle class existence—this combined with my desire to support my family, at times makes me feel like I don't have the means to pursue my dreams. Playing Real Lives 2010 forced me to acknowledge how silly such thoughts are. Reaching my dreams and doing what I love are far more reachable for me than for the vast majority of people in the world. Real Lives was an important game for me, because if I ever hope to help those less privileged, I must start by acknowledging how much has been given me.
Filed Under: Editorial Life Reflections
About the Author:
Drew Dixon is editor-in-chief of Game Church. He also edits for Christ and Pop Culture and writes about video games for Paste Magazine and Think Christian.
3 Responses to "The World Entire"
Steven Sukkau
It's frustrating, because a game like Real Lives seems to expose how lucky we are, but then what? What do you do with that information? Be grateful you are not them? But maybe that exposure, that frustration is all that is needed to spur me to action. And I would never taken action without first feeling empathy?
Jordan Rivas
Thanks for writing this, Drew. Fine piece. You sum up on a really important point about perspective, and appreciation. I haven't played Real Lives 2010 but it sounds like it offers a broad, but cold view that can trigger empathy. We so often find a way to empathy with more personal depictions, so I think this title may be unique in that way (statistics rarely cause empathy).
Drew Dixon
Thanks Jordan and Steven–glad you liked it. I would say the game is definitely worth picking up. My wife and I have had some pretty interesting discussions about the "lives" of my various characters. I think the game sorta invite you to fill in some of the gaps and in that sense is one of the few edutainment games that I have found particularly compelling.
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Random nonsense by Joe LeVasseur
Remember last year the satellite that was supposed to be snatched from the air with helicopters equipped with special hooks- but smashed into the desert? It turns out it's an example of how things can go wrong even after being tested to death.
The probable cause is that two g-switches (Accelerometer switches.) were installed upside-down. Before you blame the guy who assembled the PCB- he followed the design to the letter. In other words- Lockheed supplied the faulty design.
NASA link one
NASA link two
It would seem to be fairly easy to devise a non-destructive test to verify that the circuit would fire when hit with force from the appropriate direction.
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Limon is characterized by bright green frosty buds. It has a sour, pine flavor on the inhale with an almost citrusy bouquet. Exhale and taste the earthy, petrol funk. An uplifting smoke, potent and long lasting. Click here for pricing!
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