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The pathogenic yeast Cryptococcus neoformans causes life-threatening meningoencephalitis in individuals suffering from HIV/AIDS. The cyclic-AMP/protein kinase A (PKA) signal transduction pathway regulates the production of extracellular virulence factors in C. neoformans, but the influence of the pathway on the secretome has not been investigated. In this study, we performed quantitative proteomics using galactose-inducible and glucose-repressible expression of the PKA1 gene encoding the catalytic subunit of PKA to identify regulated proteins in the secretome.
The proteins in the supernatants of cultures of C. neoformans were precipitated and identified using liquid chromatography-coupled tandem mass spectrometry. We also employed multiple reaction monitoring in a targeted approach to identify fungal proteins in samples from macrophages after phagocytosis of C. neoformans cells, as well as from the blood and bronchoalveolar fluid of infected mice.
We identified 61 secreted proteins and found that changes in PKA1 expression influenced the extracellular abundance of five proteins, including the Cig1 and Aph1 proteins with known roles in virulence. We also observed a change in the secretome profile upon induction of Pka1 from proteins primarily involved in catabolic and metabolic processes to an expanded set that included proteins for translational regulation and the response to stress. We further characterized the secretome data using enrichment analysis and by predicting conventional versus non-conventional secretion. Targeted proteomics of the Pka1-regulated proteins allowed us to identify the secreted proteins in lysates of phagocytic cells containing C. neoformans, and in samples from infected mice. This analysis also revealed that modulation of PKA1 expression influences the intracellular survival of cryptococcal cells upon phagocytosis.
Overall, we found that the cAMP/PKA pathway regulates specific components of the secretome including proteins that affect the virulence of C. neoformans. The detection of secreted cryptococcal proteins from infected phagocytic cells and tissue samples suggests their potential utility as biomarkers of infection. The proteomics data are available via ProteomeXchange with identifiers PXD002731 and PASS00736.
Cryptococcus neoformans is an opportunistic, yeast-like fungus that is a significant threat to immunocompromised individuals such as patients with HIV/AIDS [1, 2]. The ability of C. neoformans to cause disease depends on the production of virulence factors including a polysaccharide capsule, melanin deposition in the cell wall, the ability to grow at 37 °C, and the secretion of extracellular enzymes [3–8]. Extracellular enzymes with roles in virulence include phospholipases, which hydrolyze ester bonds and aid in the degradation and destabilization of host cell membranes and cell lysis, and urease, which hydrolyzes urea to ammonia and carbamate, inducing a localized increase in pH [9–12]. Proteinases may also cause tissue damage, provide nutrients to the pathogen and facilitate migration to the central nervous system [13–15]. In general, the secretion of extracellular enzymes is important for fungal survival within the host but a comprehensive investigation of the secretome and its regulation by the cyclic-AMP/Protein Kinase A (PKA) signal transduction pathway has not been performed for C. neoformans.
The cAMP/PKA pathway regulates capsule production, melanin formation, mating, and virulence in C. neoformans [16–20]. Components of the pathway include a Gα protein (Gpa1), adenylyl cyclase (Cac1), adenylyl cyclase-associated protein (Aca1), a candidate receptor (Gpr4), phosphodiesterases (Pde1 and Pde2), and the PKA catalytic (Pka1, Pka2) and regulatory (Pkr1) subunits. In response to environmental signals, including exogenous methionine and nutrient starvation, the G-protein coupled receptor (GPCR), Gpr4 undergoes a conformational change to activate Cacl and subsequently stimulate the production of cAMP. Mutations in genes encoding the Gpa1, Cac1, Aca1, and Pka1 proteins result in reduced formation of capsule and melanin, as well as sterility and attenuated virulence in a mouse model of cryptococcosis [16, 21]. In particular, Pka1 is a key regulator of virulence in C. neoformans. In contrast, disruption of the gene encoding Pkr1 results in enlargement of the capsule and hypervirulence .
Previous transcriptional profiling experiments compared a wild-type strain with pka1Δ and pkr1Δ mutants of C. neoformans, and identified differences in transcript levels for genes related to cell wall synthesis, transport (e.g., iron uptake), the tricarboxylic acid cycle, and glycolysis . Differential expression patterns were also observed for genes encoding ribosomal proteins, stress and chaperone functions, secretory pathway components and phospholipid biosynthetic enzymes. Specifically, loss of PKA1 influenced the expression of genes involved in secretion, and Pka1 was hypothesized to influence capsule formation by regulating expression of secretory pathway components that control the export of capsular polysaccharide to the cell surface. Additionally, the secretion inhibitors brefeldin A, nocodazole, monensin, and NEM reduced capsule size, a phenotype similar to that observed in a pka1 mutant . In general, the mechanisms and components required for the export of capsule polysaccharide and other virulence factors in C. neoformans are poorly understood. Beyond the role of PKA, other studies have examined exocytosis functions (Sec6, Sec14), the secretion of phospholipases, and the involvement of extracellular vesicles [23–28]. Additionally, O’Meara et al. (2010) recently demonstrated that PKA influences capsule attachment via phosphorylation of the pH-responsive transcription factor Rim101, a key regulator of cell wall functions.
The role of PKA in secretion in C. neoformans has also been examined with strains carrying galactose-inducible and glucose-repressible versions of PKA1 and PKR1 constructed by inserting the GAL7 promoter upstream of the genes . Elevated Pka1 activity, stimulated by growth of the P GAL7 ::PKA1 strain in galactose-containing media, was found to influence capsule thickness, cell size, ploidy, and vacuole enlargement . The authors also showed that Pka1 activity was required for wild-type levels of melanization and laccase activity, and influenced the correct localization of laccase. The ability to regulate expression of PKA1 and, subsequently, the activity of Pka1, is a powerful tool for investigating the mechanisms of its influence on the secretion of virulence factors and secretory pathway components.
In this study, we used the strain with galactose-inducible and glucose-repressible expression of PKA1 to investigate the influence of Pka1 on the secretome using quantitative proteomics. We identified 61 different secreted proteins and found that Pka1 regulated the extracellular abundance of five. These proteins included three enzymes (α-amylase, acid phosphatase, and glyoxal oxidase), the Cig1 protein (cytokine-inducing glycoprotein) associated with virulence and heme uptake, and a novel protein containing a carbohydrate-binding domain (CNAG_05312). We also observed a change in the secretome profile under Pka1-inducing conditions from proteins involved primarily in catabolic and metabolic processes to an expanded set that included proteins for translational regulation and the response to stress. Enrichment analysis of our Pka1-influenced secretome data compared to the whole genome showed over-representation of genes associated with a broad spectrum of processes including metabolic and catabolic processing. Although no enrichment was observed between our secretome data and the Fungal Secretome KnowledgeBase (FunSecKB), a comparison of GO terms between the data sets showed the majority of our identified proteins to be represented in the FunSecKB. Next, we exploited our secretome data using a targeted proteomics approach to identify potential biomarkers of cryptococcal infection. Multiple Reaction Monitoring (MRM) in the presence of stable isotope dilutions (SID) allows for identification and quantification of specific peptides in a sample. Specifically, we were able to identify Pka1-regulated proteins of C. neoformans in host samples including blood, bronchoalveolar lavage fluid, and infected macrophage lysates. Overall, our study reveals that the cAMP/PKA pathway regulates specific components of the secretome including the Cig1 and Aph1 proteins that contribute to virulence in C. neoformans.
Given the virulence defect of a pka1 mutant, we hypothesized that Pka1 influences the secretion of proteins associated with the virulence and survival of C. neoformans in the host. To test this idea, we quantitatively identified proteins secreted by C. neoformans in the context of regulated expression of PKA1. For our initial analysis, we collected supernatant cultures of WT and P GAL7 ::PKA1 strains grown under Pka1-repressed (glucose) and Pka1-induced (galactose) conditions at 16, 48, 72, and 120 h post-inoculation (hpi), and analyzed the samples using quantitative mass spectrometry. The analysis of these supernatant samples resulted in the identification of 164 (54 quantifiable) and 207 (83 quantifiable) proteins under Pka1-repressed and Pka1-induced conditions, respectively (see Additional file 1: Table S1; Additional file 2: Table S2). As shown in Table 1, 23 proteins were identified and quantified under Pka1-repressed and Pka1-induced conditions at the specified time-points. We found that none of the changes in protein abundance between the two conditions were statistically significant (p > 0.05) and therefore, concluded that Pka1 did not influence the abundance of any of the observed proteins under the conditions tested. However, upon comparison of the unique proteins identified under either Pka1-repressed or Pka1-induced conditions, using Gene Ontology (GO) term biological classifications at all time points, we were able to observe overall changes in the secretome profiles under the influence of Pka1 (Fig. 1). Additional differentially expressed proteins may be present in the samples, but we were unable to measure their abundance and they were therefore not included for further analysis. Under Pka1-repressed conditions, the majority of secreted proteins were associated with catabolic and metabolic (33 %), unknown (20 %), and hypothetical (20 %) processes (totaling 73 %), with additional proteins associated with transport (8 %), oxidation-reduction processes (4 %), dephosphorylation (4 %), proteolysis (4 %), glycolysis (4 %), and regulation of transcription (3 %). Conversely, a change in the secretome profile was observed under the Pka1-induction condition. Here, we again observed the majority of proteins to be associated with catabolic and metabolic (26 %), unknown (19 %), and hypothetical (17 %) processes (totaling 62 %). A slight decline was found for proteins associated with transport (from 8 to 6 %), oxidation-reduction processes (from 4 to 3 %), dephosphorylation (from 4 to 2 %), proteolysis (from 4 to 3 %), and regulation of transcription (from 3 to 0 %). However, a greater emphasis was found for proteins associated with glycolysis (from 4 to 6 %), response to stress (from 0 to 8 %), translation (from 0 to 7 %), and nucleosome assembly (from 0 to 3 %). Although, our secretome analysis at specific times did not identify Pka1-regulated proteins, a change toward the secretion of proteins for glycolysis, translational regulation, nucleosome assembly, and the response to stress was observed upon induction of PKA1 expression.
Given that we identified secreted proteins from strains with modulated Pka1 activity, but did not observe any proteins whose abundance was directly regulated by Pka1, we extended our analysis to examine protein secretion at an intermediate time point of 96 hpi, and we used an alternative, less stringent method for protein precipitation (EtOH/acetate). We chose an end-point collection time of 96 hpi based on our coverage of a range of other time points in the previous analysis and because this time was sufficient for the culture to reach stationary phase and to accumulate proteins in the extracellular environment. Additionally, because we did not observe changes in protein abundance under regulation of Pka1 following the time-point analysis, we used the alternative protein precipitation method in an attempt to obtain a more comprehensive view of the secretome. We collected supernatant cultures of WT and P GAL7 ::PKA1 strains grown under Pka1-repressed (glucose) and Pka1-induced (galactose) conditions at 96 hpi and analyzed the samples using quantitative mass spectrometry. Similar trends in protein abundance were observed for the majority of proteins in both experimental approaches (EtOH and TCA/acetone precipitation) (see Additional file 3: Table S3) . Although the variability of the time-point analysis was relatively high, the reproducibility observed from the end-point analysis suggested that collecting the samples at different time-points impacted the protein abundance and contributed to the observed variability. This impact may be associated with culture sampling, as well as changes in capsule production during the early- to mid-log growth phases of the fungal cultures . We identified 61 proteins under Pka1-repressed conditions of which 34 were successfully dimethyl-labeled and quantified (Table 2; see Additional file 4: Table S4). These 34 proteins covered a broad spectrum of biological classifications (17 categories) for GO terms, including proteins associated with catabolic and metabolic processes, ubiquitination, transport, dephosphorylation, glycolysis, oxidation-reduction, translation, proteolysis, and the response to stress. Under Pka1-induced conditions, we identified 38 proteins, of which 21 were successfully dimethyl-labeled and quantified (Table 3; see Additional file 5: Table S5). These 21 proteins covered 11 biological classifications for GO terms and included proteins associated with catabolic and metabolic processes, along with ubiquitination, transport, dephosphorylation, oxidation-reduction, proteolysis, and the response to stress. In total, 17 proteins were present under both Pka1-repressed and Pka1-induced conditions. A comparison of changes in abundance under Pka1-repressed and Pka1-induced conditions of these 17 proteins revealed that five showed statistically significant differences (p-value < 0.05, Student’s t-test) in abundance in response to regulation of Pka1 (Fig. 2). We concluded that the extracellular abundance of these five proteins was influenced by PKA and we focused our subsequent analysis on these proteins. Under Pka1-induced conditions, a cytokine-inducing glycoprotein (Cig1), an α-amylase, a glyoxal oxidase, and a novel protein (CNAG_05312) each showed an increase in abundance, whereas an acid phosphatase (Aph1) showed a decrease in abundance. Taken together, these findings suggest that Pka1 regulates the extracellular abundance of specific proteins secreted by C. neoformans.
Based on our identification and quantification of 192 proteins in the secretome of C. neoformans, we next sought to classify the corresponding genes according to their GO terms of biological process, cellular component, and molecular function. Our goal was to assess whether subsets of genes showed significant over-representation relative to all genes in C. neoformans. To perform the enrichment analysis, all unique proteins identified under Pka1-repressed conditions were combined into a single data set as were proteins identified under Pka1-induced conditions. As shown in Fig. 3, the identified secreted proteins under Pka1-repressed conditions were enriched in 15 biological categories, with the most significant enrichment associated with carbohydrate metabolic process, catabolic process, generation of precursor metabolites and energy, organic substance metabolic process, and primary metabolic process. Under Pka1-induced conditions, enrichment was only associated with the five most significantly enriched categories under Pka1-repressed conditions. Classification by cellular components showed the most significant enrichment associated with the cytoplasm under both conditions, which may be an artifact of the classification process or indicative of the location of protein synthesis (see Additional file 6: Figure S1), whereas classification by molecular function showed no enrichment.
Our gene sets were also compared to all reported secreted proteins in the Fungal Secretome Knowledge Base (FunSecKB) for C. neoformans strain JEC21 [31, 32]. The analysis showed no significant enrichment; however, similarities among the identified GO terms were observed (Fig. 4). Forty-seven GO term categories were shared between the FunSecKB and our identified proteins under Pka1-repressed and Pka1-induced conditions; the greatest number of proteins being associated with metabolic processes. Twenty-five categories were represented only in our secretome data, and one category (GO:0009607; response to biotic stimulus) was represented only in the FunSecKB. Upon comparison of GO term categories for cellular components, 16 categories were shared between the FunSecKB and our identified proteins under Pka1-repressed and Pka1-induced conditions; the greatest number of proteins being associated with the cell, cytoplasm, and intracellular categories (see Additional file 7: Figure S2). Upon comparison of GO term categories for molecular function, 17 categories were shared between the FunSecKB and our identified proteins under Pka1-repressed and Pka1-induced conditions; the greatest number of proteins associated with binding as well as enzyme activity (see Additional file 8: Figure S3). Taken together, the enrichment analysis of our secretome data under modulation of Pka1 activity compared to the whole genome showed over-representation of genes associated with a broad spectrum of processes including metabolic and catabolic processing. Although no enrichment was observed between our secretome data and the FunSecKB, a comparison of GO terms between the data sets showed all but one of our identified proteins to be represented in the FunSecKB.
We next examined the secreted proteins, under modulation of Pka1 activity, for the presence of predicted signal peptides and GPI anchors. Specifically, we used SignalP 4.1, Signal-3 L, and Phobius for the prediction of protein extracellular location based on the presence or absence of N-terminal signal peptides. The presence of a signal peptide suggests conventional secretion versus potential non-conventional export if a signal peptide is absent. Additionally, we used GPI-SOM to predict the presence or absence of a GPI-anchor on proteins, indicative of plasma membrane association, which may or may not be capable of dissociation and subsequent protein secretion. Of the 61 proteins used for this analysis, 14 had both an N-terminal signal peptide and a GPI-anchor protein, 17 had only an N-terminal signal peptide, one had a GPI-anchor but no N-terminal signal peptide, and 29 proteins did not have an N-terminal signal peptide or a GPI-anchor (Table 4). Taken together, these results suggest that C. neoformans may employ a non-conventional secretory pathway for regulation of part of its secretome, including potential protein secretion via vesicle export .
Based on our identification and quantification of five secreted proteins regulated by Pka1 in C. neoformans, we evaluated whether transcript levels were also influenced by Pka1 regulation and whether there was a correlation with the observed regulation of protein abundance. Specifically, we performed qRT-PCR on RNA collected at 16 and 96 hpi from cells grown in Pka1-repressed and Pka1-induced conditions for the WT and P GAL7 ::PKA1 strains, and compared the observed values to our quantitative proteomic results at 96 hpi. Figure 5 summarizes the RNA expression levels at 16 hpi and 96 hpi and protein abundance at 96 hpi for Cig1, the acid phosphatase Aph1, an α-amylase, a glyoxal oxidase, and a novel protein (CNAG_05312). Cig1 and the novel protein both showed down-regulation of their transcripts under Pka1-repressed conditions at 16 and 96 hpi, followed by minimal or slight up-regulation with induced Pka1 activity. α-Amylase and glyoxal oxidase showed an initial peak in transcript levels at 16 hpi, followed by minimal change or a decrease in RNA levels at 96 hpi under Pka1-repressed conditions, and the transcript levels decreased in response to Pka1 induction. Acid phosphatase showed elevated transcript levels upon PKA1 repression at both time points, compared to a drop in RNA levels at 16 hpi or no change at 96 hpi upon induction of PKA1. In general, Pka1 appears to positively regulate the transcript levels of Cig1 and the novel protein (CNAG_05312), and to negatively regulate the transcript levels of the other three proteins. Taken together, our results suggest that although Pka1 activity influences the transcript levels and extracellular abundance of the five proteins, a correlation between transcript and protein levels was not always observed, and this was particularly notable for glyoxal oxidase. The differences may indicate additional levels of potential influence of Pka1 beyond transcriptional regulation, including differences in mRNA versus protein stability, the timing of expression and the regulation of protein export. For example, more detailed studies will be needed to examine the timing of intracellular and extracellular accumulation of the glyoxal oxidase protein relative to transcription of the gene.
Based on our identification of five Pka1-regulated proteins, including two with roles in virulence, we hypothesized that these proteins would be secreted during infection and that they might be potentially useful biomarkers of cryptococcosis. To test this idea, we used Multiple Reaction Monitoring (MRM), a powerful and targeted proteomics approach for the relative quantitative measurement of target proteins. In the presence of an internal standard, a stable isotope-labeled peptide, the amount of natural protein can be measured by comparing the signals to the labeled species. The isotopically labeled, proteotypic peptides terminate with C-terminal heavy arginine or lysine (C-term Arg U-13C6;U-15N4 or Lys U-13C6;U-15N2). In principle, the stable isotopes have the same physiochemical properties as the natural peptides and only differ by mass resulting in co-elution of the peptides. However, studies have suggested that in the presence of complex biological samples, such as blood or serum, the retention times between the peptides can shift, impacting the co-elution patterns . We specifically applied MRM to detect Cig1, Aph1, glyoxal oxidase, α-amylase, and the novel protein (CNAG_05312) in samples from a macrophage-like cell line and from infected mice.
The samples from the J774A.1 macrophage-like cell line came from cells inoculated with WT and P GAL7 ::PKA1 strains under Pka1-repressed (DMEM medium supplemented with glucose) and Pka1-induced (DMEM medium supplemented with galactose) conditions. Intracellular uptake at 2 hpi showed a significant difference in the number of colony forming units (CFUs) per macrophage between the WT and P GAL7 ::PKA1 strains under Pka1-repressed conditions, but not under induced conditions (Fig. 6a). This difference is most likely due to the absence of the capsule for the Pka1-repressed cells, a phenotype that enhances phagocytosis. By 24 hpi, rates of intracellular fungal cells per macrophage were significantly different for WT and P GAL7 ::PKA1 strains under both conditions (Fig. 6c). Specifically, intracellular rates of infection at 24 hpi in repressed conditions were 11.49 ± 2.11 % for the WT and 55.67 ± 12.76 % for P GAL7 ::PKA1 strains. However, intracellular rates under induced conditions were 9.06 ± 2.91 % for WT and 1.97 ± 0.82 % for P GAL7 ::PKA1 strains. Importantly, intracellular uptake rates showed no differences between WT, P GAL7 ::PKA1, and the pka1Δ strains under controlled growth conditions (DMEM – high glucose (0.45 %)) at 2 and 24 hpi (Fig. 6b, d). These results indicate that modulation of PKA1 expression influences the intracellular survival of cryptococcal cells.
MRM on macrophage lysates infected with fungal cells at 24 hpi identified the Pka1-regulated and secreted proteins α-amylase and glyoxal oxidase in both induced and repressed conditions. Figure 7 shows representative chromatographic co-elution patterns of the isotopically-labeled and natural peptides, which allowed for relative quantification of peptides in the replicates of the experiment. For both enzymes, the highest amount of protein was detected in the WT strain in DMEM medium under Pka1-repressed conditions, whereas the P GAL7 ::PKA1 strain under Pka1-induction showed the lowest amount of secreted protein. This observation may be associated with reduced intracellular rates of the P GAL7 ::PKA1 strain due to the presence of an enlarged capsule. Overall, we were able to detect 29.8 ± 37.0 fmol of α-amylase and 149.1 ± 130.0 fmol of glyoxal oxidase in 5 μg of total protein from the macrophage lysate following the uptake of P Gal7 ::PKA1 under Pka1-induced conditions at 24 hpi.
The samples from infected mice included BAL and blood from animals inoculated with the WT strain. Three mice were selected for each type of in vivo analysis based on previous studies of cryptococcosis [34–37]. Representative chromatograms of isotopically-labeled and natural peptides detected in mouse BAL are presented in Fig. 8. The MRM analysis identified Cig1, α-amylase, glyoxal oxidase, and the novel protein (CNAG_05312) in BAL following infection with WT cells. In 5 μg of total protein, glyoxal oxidase was the most abundant protein with detection at 779.5 ± 436.1 fmol, followed by the novel protein (CNAG_05312) at 451.0 ± 90.5 fmol, Cig1 at 291.3 ± 54.5 fmol, and α-amylase with the lowest abundance at 40.1 ± 9.4 fmol. Lastly, we were able to detect Cig1, glyoxal oxidase, and the novel protein (CNAG_05312) in blood. Representative chromatograms of the isotopically-labeled and natural peptides detected in mouse blood are presented in Fig. 9. Again, glyoxal oxidase was the most abundant protein detected at 319.4 ± 272.7 fmol, followed by Cig1 at 62.0 ± 17.4 fmol, and the novel protein (CNAG_05312) at 3.1 ± 3.8 fmol in 5 μg of total protein. Aph1 levels were below the limit of detection in all samples. Taken together, our targeted proteomics approach identified and quantified the Pka1-regulated secreted proteins as potential biomarkers following host challenge with cryptococcal cells.
The secretion of extracellular enzymes and virulence-associated factors is important for the proliferation and survival of pathogens in the host environment. For the pathogenic yeast C. neoformans, virulence depends to a large extent on the export of polysaccharide to form a capsule, as well as targeted delivery of laccase to the cell wall for deposition of melanin, and secretion of extracellular enzymes [23, 24, 28, 38]. The cyclic-AMP/Protein Kinase A signal transduction pathway plays a key role in regulating these processes but the underlying mechanisms remain to be understood in detail [16, 17]. We therefore used a P GAL7 ::PKA1 strain under Pka1-repressed and Pka1-induced conditions in this study to investigate the influence of Pka1 on the secretome of C. neoformans. Quantitative proteomics allowed us to identify 61 different proteins in the secretome including a subset of five whose abundance was regulated by Pka1. These five proteins include a cytokine-inducing glycoprotein (Cig1), an α-amylase, a glyoxal oxidase, an acid phosphatase (Aph1), and a novel protein (CNAG_05312). We also observed a change in the secretome profile upon induction of PKA1 expression thus establishing a view of the impact of PKA activity on the extracellular protein composition. In general, this analysis highlighted the enrichment of Pka1-regulated biological processes in the secretome, revealed potential targets for conventional and non-conventional modes of secretion, and provided candidate biomarkers for investigating cryptococcosis.
Our analysis revealed a change in the abundance of secreted C. neoformans proteins associated with glycolysis, translational regulation, nucleosome assembly, and stress response over a time course from 16 to 120 h. We speculate that some of these proteins may result from packaging in vesicles known to transit through the cell wall and accumulate in the extracellular environment [24, 39]. In this case, modulation of PKA activity may indirectly influence the proteome of vesicles as a reflection of an impact on the intracellular proteome. This idea is supported by our observed influence of PKA1 modulation on the abundance of the translation machinery because ribosomal proteins, in particular, are abundant in extracellular vesicles . It is also well known that PKA influences the transcription of ribosomal protein genes in other organisms and this influence is conserved in C. neoformans [22, 40]. Our analysis of the intracellular proteome also revealed suppression of ribosomal cellular protein abundance upon induction of Pka1 (Geddes et al., unpublished data). We also observed a connection between Pka1 activation and the abundance of glycolytic proteins. This is interesting in light of previous reports demonstrating the importance of glycolysis for virulence and the persistence of C. neoformans in the cerebral spinal fluid . These findings are consistent with a previous analysis of the transcriptome, which showed that Pka1 influences the levels of transcript for genes involved in glycolysis . Furthermore, the observed influence of Pka1 induction on the secretion of proteins associated with stress response is consistent with observed Pka1 regulation at the transcriptional level. In this context, we identified a heat shock protein 70 (Hsc70-4), which is associated with the response to stress and which was previously localized to the cell surface of C. neoformans . The observed connection between the stress response and Pka1 induction may indicate coordination for facilitation of fungal survival and proliferation during colonization of vertebrate hosts.
The influence of PKA on the abundance of the mannoprotein Cig1 is of particular interest because we previously showed that its transcript is one of the most abundant in cells grown in low iron medium . In addition, the protein is important for iron acquisition from heme and virulence in C. neoformans . We found that the extracellular abundance of Cig1 increased upon induction of Pka1 and that transcript levels and protein abundance were well correlated. CIG1 is positively regulated by the pH-responsive transcription factor Rim101, which in turn is activated by the cAMP/PKA pathway . Therefore, the regulation of CIG1 mRNA and Cig1 protein levels observed upon induction of Pka1 likely reflect regulation by Rim101. This finding is consistent with recent discoveries that Rim101 controls cell wall composition and capsule attachment via an influence on the expression of cell wall biosynthetic genes [46, 47].
In general, a number of proteins associated with cell wall synthesis and integrity, pathogenesis and the immune response were prominent in the secretome of C. neoformans upon modulation of PKA1 expression. These proteins included an endo-1,3(4)-β glucanase and a 1,3-β-glucanosyltransferase, both of which have been previously identified in studies of the extracellular proteomes of C. neoformans and other fungal pathogens such as Histoplasma capsulatum [28, 48–51]. Endo-1,3(4)-β glucanase is located in the surface layers of the cell wall or in the capsule and has roles in metabolism, autolysis, and cell separation [50, 52]. The 1,3-β-glucanosyltransferase is described as a glycolipid protein anchored to the cell membrane in yeasts and may have a role in virulence . Our proteomic analysis also identified chitin deacetylases associated with the formation of chitin and cell wall integrity, and the enzyme laccase, which is responsible for melanin deposition in the cell wall and influences cryptococcal virulence [28, 51, 54–57]. These findings are consistent with our previous transcriptomic analysis, which revealed an influence of PKA on the expression of cell wall associated genes .
We also identified a novel protein (CNAG_05312) with a pattern of mRNA and protein regulation by Pka1 activity that was quite similar to that of Cig1. This novel protein contains a predicted carbohydrate-binding domain and was annotated as a macrophage-activating glycoprotein (reminiscent of the cytokine-inducing glycoprotein designation of Cig1). These observations suggest that further investigation is warranted for this protein in the context of iron acquisition and virulence. This idea is reinforced by the finding that Rim101 also positively regulates expression of the CNAG_05312 gene . Interestingly, the CNAG_05312 gene is also regulated at the transcript level by the transcription factor Gat201 that, like Pka1, influences capsule size, virulence, and uptake by macrophages [58, 59]. Considering these similar phenotypes, it is possible that Gat201 and Pka1/Rim101 both regulate the expression of the CNAG_05312 protein and subsequently influence the activation of macrophages during infection. Overall, our investigation of the secretome reinforced connections between modulation of Pka1 activity, Rim101 and cell wall integrity, and it revealed an impact of PKA on the extracellular abundance of proteins with known (Cig1) and potential (the novel CNAG_05312 protein) influences on virulence.
Pka1 also positively regulated the abundance in the secretome of an α-amylase and a glyoxal oxidase which were previously identified in the extracellular proteome of C. neoformans [28, 51]. Amylases are associated with carbohydrate metabolism, particularly starch degradation for energy production . In C. neoformans, the secretion of amylases in the PKA-regulated strains was reported previously and we were able to measure and confirm α-amylase activity in the extracellular medium . Glyoxal oxidases are extracellular H2O2-producing enzymes associated with cellulose metabolism . There is evidence that glyoxal oxidase activity is involved in filamentous growth and pathogenicity of Ustilago maydis, as well as fertility in Cryptococcus gattii [61, 62]. A similar pattern in response to PKA1 expression was observed upon comparison of the transcript and protein levels for both the α-amylase and the glyoxal oxidase. A direct correlation between transcript levels and protein abundance was not as evident as for Cig1. This could potentially be due to post-transcriptional regulation, differences in mRNA and protein half-lives and issues with timing . It is also possible that PKA may regulate additional processes to influence extracellular protein abundance, such as the activity of the secretory pathway. Overall, the secretome data revealed a new connection between PKA regulation and the α-amylase and glyoxal oxidase enzymes, and this discovery indicates that further analysis of their potential roles in virulence is warranted.
The extracellular abundance of the acid phosphatase Aph1 and its transcript levels were negatively regulated by induction of PKA1 expression thus revealing an opposite pattern of regulation compared with the other four genes. Phosphatases have been predicted to have roles in cell wall biosynthesis, cell signaling, phosphate scavenging, and in adhesion of C. neoformans to epithelial cells [24, 28, 64–67]. The APH1 gene was recently characterized and its expression was found to be induced by phosphate limitation; the Aph1 protein was also the major conventionally secreted acid phosphatase in C. neoformans . Aph1 was also shown to hydrolyze a variety of substrates to potentially scavenge phosphate from the environment, and an aph1 deletion mutant had a slight virulence defect in both Galleria mellonella and mouse models of cryptococcosis. The latter phenotype is consistent with our recent study showing that a high affinity phosphate uptake system is required for growth on low-phosphate medium, for formation of the virulence factors melanin and capsule, for survival in macrophages, and for virulence in mice . This study also revealed that defects in PKA influence the growth of C. neoformans on phosphate-limited medium. Our discovery of PKA regulation of Aph1 abundance in the secretome therefore further reinforces a connection between phosphate acquisition and PKA regulation associated with virulence.
Our profiling of the secretome upon modulation of Pka1 activity confirmed the presence of previously identified extracellular and vesicular proteins, including those associated with virulence and fungal survival within the host, as well as novel secreted proteins. We identified the classically secreted C. neoformans protein, laccase, associated with fungal virulence, but other proteins such as urease and phospholipase B were not identified in our study. Their absence could be attributed to growth conditions, precipitation methods, supernatant collection times, and relative abundance in the secretome. A recent proteome study that removed free capsular polysaccharide from the extracellular environment identified 105 secreted proteins and a direct comparison with our study showed an overlap of 52 % . Previous investigation of the proteins in extracellular vesicles of C. neoformans also showed an overlap of nearly 56 % with proteins identified in our study [24, 39]. This overlap is primarily associated with proteins not typically expected in the secretome. For example, ATP subunits/carriers, translation elongation factor, actin, and multiple ribosomal proteins were identified and their presence was attributed to packaging in extracellular vesicles, and not necessarily due to direct secretion. In the absence of an N-terminal signal peptide, proteins may be exported via non-conventional secretion. This may include the use of membrane-bound, extracellular vesicles capable of traversing the cell wall, the possible fusion of multi-vesicular bodies with the plasma membrane, or the capture of cytosolic material to form vesicles (blebbing), as discussed above [23, 24, 69–72]. Taken together, our profile of secreted proteins in C. neoformans is in agreement with previous secretome studies. However, our ability to modulate Pka1 activity provides an opportunity to identify novel proteins in the extracellular environment as well as identify proteins specifically regulated by Pka1. This approach led to the unique identification of the novel secreted protein (CNAG_05312) that was specifically associated with modulation of Pka1 activity and not found in other proteomic studies.
Biomarkers are indicators of normal or pathogenic processes as well as the efficacy of therapy . In this regard, targeted detection of secreted cryptococcal proteins provides an opportunity to identify potential biomarkers for early diagnosis of infection and to monitor antifungal therapy. Early and rapid diagnosis remains limited for systemic fungal infections, such as those caused by Candida and Aspergillus species, as well as C. neoformans and C. gattii . Biomarkers of infection by specific fungal species would therefore be valuable for identification and for precise measurements of fungal burden. A recent study using the presence of the cell wall component galactomannan in BAL as a diagnostic tool for invasive fungal disease highlights an opportunity for biomarker discovery in fungal pathogens . Additionally, the use of targeted proteomics (and MRM in particular) is a novel approach to study the secretion of virulence factors in C. neoformans, particularly in the context of signaling functions like PKA that sense conditions relevant to the host environment.
The secreted proteins that we identified to be regulated in abundance by Pka1 provide an opportunity to develop diagnostic biomarkers that are also informative about signaling via the cAMP/PKA pathway in vitro and during infection. For example, Cig1 is an important candidate biomarker given its abundance in iron-starved cells and its role in virulence through iron acquisition and uptake. Our ability to detect Cig1 in the blood and BAL fluid of infected animals confirms its expression and establishes the protein as a potential biomarker. These findings may also indicate a role for Cig1 in iron uptake in these environments although, interestingly, we did not detect Cig1 in macrophage lysates. Based on our observed differences in intracellular replication, Pka1 seems to impact the intracellular environment of macrophages. In this regard, we did detect the glyoxal oxidase and α-amylase proteins by MRM in macrophages containing cryptococcal cells. Expression of these proteins has not previously been reported during interactions with macrophages, although the production of H2O2 and induction of oxidative stress via glyoxal oxidase could potentially influence intracellular survival. It is known that oxidative stress induces autophagy in macrophages and can impair phagocytic activity [76, 77]. Additionally, loss of an α-amylase in H. capsulatum attenuated the ability of the fungus to kill macrophages and to colonize murine lungs . This influence appeared to be related to the ability to produce α-(1,3)-glucan. The regulation of glyoxal oxidase and α-amylase by Pka1 activity and their detection in macrophage lysates suggests that it would be interesting to examine the roles of these enzymes in intracellular survival and virulence. Our approach with MRM is also informative about tissue specific expression of fungal proteins during disease. In addition to the examples described above, we found that colonization of murine lungs resulted in secretion of α-amylase, glyoxal oxidase and the novel protein from gene CNAG_05312. The novel protein was also found in blood and, given its similar regulation with Cig1 these results suggest future studies on the role of this protein in iron acquisition and virulence.
In this study we characterized the overall impact of PKA1 modulation on the secretome and discovered five proteins regulated by Pka1. The identified proteins had known roles associated with cell wall functions, fungal survival within the host, and virulence. Our identification of a novel protein with potential roles in iron uptake and virulence also suggested a previously unknown connection between Pka1 and Gat201. We were also able to detect Pka1-regulated secreted proteins in biological samples as potential biomarkers, providing a new opportunity for diagnosing fungal infection and monitoring disease progression.
The C. neoformans var. grubii wild-type strain H99 (WT) and the P GAL7 ::PKA1 strain with galactose-inducible/glucose repressible expression of PKA1 were used for this study [16, 29]. The strains were maintained on yeast extract peptone dextrose (YPD) medium (1 % yeast extract, 2 % peptone, 2 % dextrose, and 2 % agar). For studies involving regulation of PKA1, cells of the WT and regulated strains were pre-grown overnight with agitation at 30 °C in YPD broth, transferred to yeast nitrogen base medium with amino acids (YNB, Sigma-Aldrich) and incubated overnight with agitation at 30 °C. Cell counts were performed and 5 x 107 cells/ml were transferred to Minimal Medium (MM) (29.4 mM KH2PO4, 10 mM MgSO4 • 7H2O, 13 mM glycine, 3 μM thiamine, 0.27 % carbon source) containing either glucose (MM + D) or galactose (MM + G). For end-point studies, cells were incubated with agitation at 30 °C in MM + D or MM + G for 96 h; for time-course studies, cells were incubated with agitation at 30 °C in MM + D or MM + G for 16, 48, 72, and 120 h. Time points were selected based on previous studies on the timing of protein secretion as well as the analysis of proteins in extracellular vesicles of C. neoformans, which used samples collected at 48 and 72 h of growth [23, 24, 64]. Samples were collected in triplicate for analysis.
To collect supernatant samples, cells were removed by centrifugation at 3,500 rpm for 15 min at 4 °C and the culture medium was transferred to new tubes; this step was repeated four times until all cell debris had been removed. Supernatant samples were kept on ice and total protein concentration was measured by a BCA-Protein-assay (Pierce). Ultrapure bovine serum albumin was used as a calibration standard. In addition to using two approaches for protein precipitation as described below, we also used a combination of sample collection time points (time-point and end-point analyses) to maximize protein detection and obtain a comprehensive view of the secretome. The first approach involved a time-course study in which a stringent trichloroacetic acid (TCA)/acetone precipitation was performed . In brief, an aliquot of culture supernatant (50 μg total protein) was mixed with five volumes of ice-cold TCA/acetone (20 %/80 % w/v) and incubated overnight at −20 °C. Precipitated proteins were collected by centrifugation at 10,000 rpm for 20 min at 4 °C. The pellet was washed four times with ice-cold acetone, air-dried and stored at −20 °C. The second approach, which was less stringent than the TCA/acetone method, was used for the end-point studies and involved ethanol (EtOH)/acetate precipitation . In brief, an aliquot of culture supernatant (50 μg total protein) was diluted with 4 volumes of absolute EtOH, 2.5 M NaCH3COO was used to bring the solution to 50 mM NaCH3COO, pH 5.0 and 20 μg of glycogen was added to the sample. Samples were vortexed and incubated at room temperature for 2 h with periodic agitation. Precipitated proteins were collected by centrifugation at 15,000 rpm for 10 min at 4 °C. The pellet was washed twice with EtOH, then air-dried and stored at −20 °C. All supernatant samples were subjected to in-solution digestion using ACS grade chemicals or HPLC grade solvents (Thermo Scientific and Sigma-Aldrich) . In brief, the precipitated protein pellet was solubilized in digestion buffer (1 % sodium deoxycholate, 50 mM NH4HCO3), incubated at 99 °C for 5 min with agitation, followed by reduction (2 mM of dithiothreitol (DTT) for 25 min at 56 °C), alkylation (4 mM of iodoacetamide (IAA) for 30 min at room temperature in the dark), and trypsinization (0.5 μg/μl of sequencing grade modified trypsin (Promega)) overnight at 37 °C. Based on our results, the TCA/acetone precipitation method appeared to be more stringent, perhaps due to more extensive washing in the protocol.
Digested peptides from supernatants were desalted, concentrated, and filtered on C18 STop And Go Extraction (STAGE) tips . Reductive dimethylation using formaldehyde isotopologues was performed to differentially label peptides from the different experimental conditions. Light formaldehyde (CH2O) and medium formaldehyde (CD2O) (Cambridge Isotope Laboratories, Andover, MA) were combined with cyanoborohydride (NaBH3CN, Sigma-Aldrich) to give a 4 Da difference for labeled peptides . Samples from the WT strain were routinely labeled with light formaldehyde, and P GAL7 ::PKA1 samples were labeled with medium formaldehyde. Briefly, eluted and dried STAGE-tip peptides were resuspended in 100 mM triethylammonium bicarbonate, and incubated in 200 mM formaldehyde and 20 mM sodium cyanoborohydride for 90 min in the dark. After labeling, 125 mM NH4Cl was added and incubated for 10 min to react with excess formaldehyde, followed by the addition of acetic acid to a pH < 2.5 to degrade sodium cyanoborohydride. For each comparison, equal amounts of labeled peptides were mixed and desalted on C18 STAGE tips.
Purified peptides were analyzed using a linear-trapping quadrupole - Orbitrap mass spectrometer (LTQ-Orbitrap Velos; Thermo Fisher Scientific) on-line coupled to an Agilent 1290 Series HPLC using a nanospray ionization source (Thermo Fisher Scientific). This includes a 2-cm-long, 100-μm-inner diameter fused silica trap column, 50-μm-inner diameter fused silica fritted analytical column and a 20-μm-inner diameter fused silica gold coated spray tip (6-μm-diameter opening, pulled on a P-2000 laser puller from Sutter Instruments, coated on Leica EM SCD005 Super Cool Sputtering Device). The trap column was packed with 5 μm-diameter Aqua C-18 beads (Phenomenex, www.phenomenex.com) while the analytical column was packed with 3.0 μm-diameter Reprosil-Pur C-18-AQ beads (Dr. Maisch, www.Dr-Maisch.com). Buffer A consisted of 0.5 % aqueous acetic acid, and buffer B consisted of 0.5 % acetic acid and 80 % acetonitrile in water. Samples were resuspended in buffer A and loaded with the same buffer. Standard 90 min gradients were run from 10 % B to 32 % B over 51 min, then from 32 % B to 40 % B in the next 5 min, then increased to 100 % B over a 2 min period, held at 100 % B for 2.5 min, and then dropped to 0 % B for another 20 min to recondition the column. The HPLC system included Agilent 1290 series Pump and Autosampler with Thermostat; temperature was set at 6 °C. The sample was loaded on the trap column at 5 μl/min and the analysis was performed at 0.1 μl/min. The LTQ-Orbitrap was set to acquire a full-range scan at 60,000 resolution from 350 to 1600 Th in the Orbitrap to simultaneously fragment the top ten peptide ions by CID and top 5 by HCD (resolution 7500) in each cycle in the LTQ (minimum intensity 1000 counts). Parent ions were then excluded from MS/MS for the next 30 s. Singly charged ions were excluded since in ESI mode peptides usually carry multiple charges. The Orbitrap was continuously recalibrated using lock-mass function . Mass accuracy included an error of mass measurement within 5 ppm and did not exceed 10 ppm.
For analysis of mass spectrometry data, centroid fragment peak lists were processed with Proteome Discoverer v. 1.2 (Thermo Fisher Scientific). The search was performed with the Mascot algorithm (v. 2.4) against a database comprised of 6,692 predicted protein sequences from the source organism C. neoformans H99 database (C. neoformans var. grubii H99 Sequencing Project, Broad Institute of Harvard and MIT, http://www.broadinstitute.org/) using the following parameters: peptide mass accuracy 10 parts per million; fragment mass accuracy 0.6 Da; trypsin enzyme specificity with 1 max missed cleavages; fixed modifications - carbamidomethyl, variable modifications - methionine oxidation, deamidated N, Q and N-acetyl peptides, dimethyl (K), dimethyl (N-term), dimethyl 2H(4) (K), and dimethyl 2H(4) (N-term), ESI-TRAP fragment characteristics. Only those peptides with Ion Scores exceeding the individually calculated 99 % confidence limit (as opposed to the average limit for the whole experiment) were considered as accurately identified. The acceptance criteria for protein identification were as follows: only proteins containing at least one unique peptide with a Mascot score > 25 were considered in the dataset. Quantitative ratios were extracted from the raw data using Proteome Discoverer. Proteome Discoverer parameters – Event Detector: mass precision 4 ppm (corresponds to extracted ion chromatograms at ±12 ppm max error), S/N threshold 1; Precursor Ion Quantifier method set for ‘2 labels’ for the formaldehyde labeled samples; Quantitation Method – Ratio Calculation – Replace Missing Quantitation Values with Minimum Intensity – yes, Use Single Peak Quantitation Channels – yes, − Protein Quantification – Use All Peptides – yes.
Experimentally determined fold changes for WT and P GAL7 ::PKA1 strains grown under Pka1-repressed (glucose-containing medium) and Pka1-induced (galactose-containing medium) conditions were converted to a log2 scale and the average fold change and standard deviation were used for analysis. A fold change of >10 was used as a cut-off limit for the time-point and end-point analyses. For the comparative analysis of the time-point samples, the statistical significance of the fold changes of the identified secreted proteins present under both Pka1-repressed and Pka1-induced conditions and at equivalent time points (i.e. 16, 48, 72, and 120 hpi) was assessed for an influence of PKA regulation using a Student’s t-test (p-value < 0.05). For the comparative analysis of the end-point samples, the statistical significance of the fold changes of the identified secreted proteins present under both Pka1-repressed and Pka1-induced conditions was evaluated using a Student’s t-test (p-value < 0.05). To confirm the statistically significant Pka1-regulated proteins identified from the end-point analysis, a multiple-hypothesis testing correction was performed on the secretome data using the Benjamini and Hochberg method with a false discovery rate of 0.05.
Proteins were characterized with Gene Ontology (GO) terms using a local installation of Blast2GO . Gene annotation data of the C. neoformans H99 reference genome were retrieved from the Broad Institute (May 2014) and a copy of the non-redundant (nr) protein database was downloaded from NCBI (May 2014) . The most current associations between the nr protein database and GO terms were retrieved in May 2014 from Blast2GO. GO terms were assigned to WT proteins and filtered using default settings of the Blast2GO pipeline . We performed GO term enrichment analyses for sets of proteins using hypergeometric tests and the Benjamini and Hochberg false discovery rate multiple testing correction (p-value < 0.05) implemented in the R packages GSEABase and GOstats. GO term categories containing singleton entries were excluded. GO categories and enrichment datasets were visualized using the R package ggplot2 . For time-point analyses, GO term classification was performed on unique proteins identified under either Pka1-repressed or Pka1-induced conditions to highlight the overall influence of Pka1 regulation on the secretome profile.
SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) was used to predict whether identified proteins were secreted based on the presence of a signal peptide. Identified protein sequences were also analyzed using Signal-3 L (http://www.csbio.sjtu.edu.cn/bioinf/Signal-3L/) and Phobius (http://phobius.sbc.su.se) to confirm results. Additionally, secreted proteins were analyzed for the presence of a glycophosphatidylinositol (GPI) anchor using GPI-SOM (http://gpi.unibe.ch).
Cells from WT and P GAL7 ::PKA1 strains were prepared for the examination of gene expression by overnight growth in YNB medium followed by dilution to 5.0 x 107 cells/ml in 5 ml of MM + D or MM + G and incubation at 30 °C with agitation for 16 and 96 h. Samples were collected in triplicate for analysis. Cells were collected at the designated time points, flash frozen in liquid N2, and stored at −80 °C. Total RNA was extracted using an EZ-10 DNAaway RNA Miniprep kit (Bio Basic) according to the manufacturer’s protocol. Complementary DNA was synthesized using a Verso cDNA kit (Thermo Scientific) and used for quantitative real-time PCR (qRT-PCR). Primers were designed using Primer3 v.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/) and targeted to the 3’ regions of transcripts. qRT-PCR primer sequences (see Additional file 9: Table S6). Relative gene expression was quantified using the Applied Biosystems 7500 Fast Real-time PCR system. Control genes CNAG_00483 (Actin) and CNAG_06699 (GAPDH) were used for normalization, and tested for statistical significance using the Student’s t-test. As a control, PKA1 RNA expression levels under Pka1-repressed and Pka1-induced conditions in the WT and P GAL7 ::PKA1 strains were also analyzed at various time points to confirm the regulated PKA expression (see Additional file 10: Figure S4).
To confirm qRT-PCR results, total RNA was isolated for the P GAL7 ::PKA1 strain grown in 50 ml of MM + D or MM + G for 16 h. Briefly, cell pellets were collected and flash frozen in liquid N2, followed by overnight lyophilization. One milliliter of buffer 1 (2 % SDS, 68 mM Na3C6H5O7, 132 mM C6H8O7, 10 mM EDTA) was added to the samples, along with 600 μl of glass beads; samples were subjected to bead beating for two, 3 min intervals at power 3 (BioSpec, Mini-Beadbeater) and subsequently stored on ice. Next, 340 μl of buffer 2 (4 M NaCl, 17 mM Na3C6H5O7, 33 mM C6H8O7) was added and samples were inverted several times and incubated on ice for 5 min. Samples were then centrifuged at 15,000 rpm for 10 min, the supernatant fraction was collected and transferred to a new tube, one volume of isopropanol was added, and samples were mixed and incubated at room temperature for 15 min. The pellet was collected following centrifugation at 15,000 rpm for 5 min, and washing of the pellet with 70 % DEPC (Diethylpyrocarbonate)-EtOH was performed. The pellet was collected, air dried, and dissolved in 20 μl of DEPC-H2O. The hybridization probes were prepared with a PCR-amplified DNA fragment of CNAG_00483 (Actin) or CNAG_00396 (PKA1) using specific primers (see Additional file 9: Table S6) and labeled with 32P using an Oligolabeling kit (Amersham Biosciences). Scanned images were analyzed using a Bio-Rad ChemiDoc MP Imaging System (see Additional file 11: Figure S5).
The survival rates of the WT, pka1Δ mutant, and P GAL7 ::PKA1 strains during incubation with macrophages were determined and lysates were prepared for protein analysis . Briefly, cells of the J774A.1 macrophage-like cell line were grown to 80 % confluence in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma) supplemented with 10 % fetal bovine serum and 2 mM L-glutamine at 37 °C and 5 % CO2. The macrophages were stimulated 1 h prior to infection with 150 ng/ml phorbol myristate acetate (PMA). Fungal cells were grown in YNB overnight at 30 °C, followed by inoculation in MM + D or MM + G at 5.0 x 107 cells/ml. Following overnight growth, the fungal cells were washed with phosphate-buffered saline (PBS, Invitrogen) and opsonized with 0.5 μg/ml of the anti-capsule monoclonal antibody 18B7 in DMEM or DMEM supplemented with 0.20 % glucose or galactose (30 min at 37 °C). Stimulated macrophages were infected with 2.0 x 105 opsonized fungal cells at a multiplicity of infection (MOI) of 1:1 for 2 h and 24 h at 37 °C and 5 % CO2. To measure fungal survival, macrophages containing internalized cryptococcal cells were washed thoroughly four times with PBS and then lysed in 1 ml of sterile dH2O for 30 min at room temperature. Lysate dilutions were plated on YPD agar and incubated at 30 °C for 48 h, at which time the resulting colony forming units (CFUs) were counted and intracellular rates of infection (%) were calculated as the ratio of the CFUs at 2 h and 24 h over the initial number of macrophages. The statistical significance of differences between WT, pka1Δ mutant, and P GAL7 ::PKA1 strains were determined by unpaired t-tests. For proteomic analysis, lysates from infected macrophage at 24 h of incubation were collected, flash frozen in liquid N2 and stored at −80 °C.
Female BALB/c mice (10–12 weeks old) obtained from Charles River Laboratories (Senneville, Ontario, Canada) were used to collect bronchoalveolar lavage (BAL) and blood samples following cryptococcal infection. Three different cultures of C. neoformans WT cells were grown overnight in YPD at 30 °C with agitation, washed in PBS and re-suspended at 1.0 x 108 cells/ml in PBS. For collection of BAL, intranasal inoculation of three mice with 100 μl of the different WT cell suspensions (1.0 x 107 cells) was performed. For collection of blood samples, intravenous inoculation of three mice with 100 μl of the WT cell suspensions (1.0 x 107 cells) was performed. Three mice were selected for the analysis based on established methods for studying fungal burden in mouse models of cryptococcosis [34–37]. At 48 hpi, the infected mice were euthanized by CO2 inhalation and 1 ml of BAL fluid and 500 μl of blood samples were collected from each mouse . Mouse lavage and blood samples were flash frozen with liquid N2 and stored at −80 °C. Mouse assays were conducted in accordance with University of British Columbia’s Committee on Animal Care (protocol A13-0093).
Macrophage lysate samples were prepared as described above, followed by trypsin in-solution digestion. Samples were collected for WT and P GAL7 ::PKA1 strains at 24 hpi in triplicate. Mouse BAL samples were prepared as described above, followed by trypsin in-solution digestion. Samples were collected at 48 hpi following WT inoculation of each of the three mice. For mouse blood samples, highly abundant proteins were removed as previously described . Briefly, proteins were precipitated by the addition of two volumes of acetonitrile and 1.0 % acetic acid, followed by centrifugation at 10,000 rpm for 5 min at 4 °C. The supernatant was collected and evaporated and the residual proteins were then subjected to trypsin in-solution digestion as described above. Samples were collected at 48 hpi following WT inoculation of each of the three mice. Following trypsin digestion, all samples were desalted, concentrated, and filtered on high-capacity C18 STAGE tips.
Skyline (v2.1) was used to build and optimize the MRM method for the relative quantification of peptides . Synthesized peptides for MRM analysis were designed in-house using the following parameters: tryptic peptides, 0 max missed cleavages, minimum of seven and maximum of 25 amino acids, excluding peptides containing Met or Cys residues (if possible) and N-terminal glutamine, hydrophobicity between 10–40 (Sequence Specific Retention Calculator, http://hs2.proteome.ca/SSRCalc/SSRCalcX.html), desirable spectral intensities (GenePattern ESPPredictor, http://www.broadinstitute.org/cancer/software/genepattern/modules), and transition settings selecting for precursor charges of 2 and 3, ion charge of 1, monitoring both b and y ions. SpikeTides labeled with stable isotopes (C-term Arg U-13C6;U-15N4 or Lys U-13C6;U-15N2) were purchased from JPT Peptide Technologies GmbH (Berlin, Germany). N-terminal Arginine (R) and Lysine (K) were labeled with a stable isotope mass of 10.008269 and 8.014199, respectively. Collision energy (CE) and fragmentor voltage (FV) for each peptide was predicted utilizing Skyline software and then confirmed experimentally . Doubly and triply charged precursor ions were optimized and three to five transitions were measured per peptide. The final MRM method included the monitoring of a total of 23 peptides, representing five proteins (see Additional file 12: Table S7). Stable isotope-labeled peptides were resuspended in 100 μl of 0.5 % acetic acid with agitation at room temperature. The peptides were further diluted and combined to result in final concentrations of 100 fmol/μl to 1 pmol/μl of each peptide. Five μl of the peptide mixture was injected into an Agilent 6460 Triple Quadrupole (Agilent) for data acquisition and peptide optimization.
MRM assays were performed on an Agilent 6460 Triple Quadrupole coupled with Agilent 1200 Series HPLC. The instrument was operated in positive electrospray ionization mode using MassHunter Workstation Data Acquisition (v.B.04.04, Agilent). Chromatography was performed on a Large Capacity Chip with 160 nl Trap, analytical column was 150 mm x 75 μm, stationary phase for both trapping and analytical columns were Zorbax-SB C-18, 300 A and 5 μm particles (Agilent). Peptides were separated using gradient elution with a stable flow of 0.30 μl/min, beginning with 97 % buffer A (97 % dH2O, 3 % acetonitrile, 0.1 % formic acid (FA)) and 3 % buffer B (10 % dH2O, 90 % ACN, 0.1 % FA) followed by a step gradient of buffer B from 3 to 80 %, which was achieved at 10.5 min. Subsequent equilibration was performed for 4.5 min at 3 % buffer B. The column was maintained at room temperature during analysis, and the samples were kept at 4-7 °C. The MS was operating in selective reaction mode using electrospray ionization in positive ion mode, with a capillary voltage of 1850 V and a source temperature of 325 °C. Cone voltage was static, collision energies and fragmentor voltages were optimized for each compound individually (see Additional file 13: Table S8). Peak identification was performed using MassHunter Qualitative Analysis (Agilent).
Quantification of natural proteins was performed using peak areas relative to the known amounts of added isotopically-labeled synthetic peptides during a multiplexed MRM run. Natural protein levels were identified in triplicate from the following matrices: WT and P GAL7 ::PKA1 macrophage lysate MM + D and MM + G collected at 24 hpi; BAL collected at 48 hpi from three different mice inoculated independently with the WT strain; and blood collected at 48 hpi from three different mice inoculated independently with the WT strain. Each biological sample was assayed independently in triplicate. Experimentally determined peak areas and the subsequent quantification values were converted to a log2 scale, and the average amount of identified peptide +/− S.D. was reported. Positive association of natural peptides to their respective isotopically-labeled peptides was determined based on co-elution patterns. For positive identification of a natural protein in a collected sample, at least one peptide with a minimum of two transitions must be identified or a minimum of two peptides with at least one transition each must be present.
Enzymatic activity was assayed for α-amylase and acid phosphatase. The assays were performed with kits for both enzymes according to the manufacturer’s protocol (BioVision Incorporated) (see Additional file 14: Figure S6). To confirm that proteins identified in the secretome were a result of secretion and not a product of cell lysis, a PCR was performed on secretome samples from Pka1-repressed conditions for WT strain at 96 hpi. Actin (CNAG_00483) and PKA1 (CNAG_00396) were used as control genes for amplification (see Additional file 15: Figure S7) .
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium  via the PRIDE partner repository  with the dataset identifier PXD002731 and the PASSEL partner repository with the dataset identifier PASS00736.
Mouse assays were conducted in accordance with University of British Columbia’s Committee on Animal Care (protocol A13-0093).
The authors thank J. Choi for strain construction, and M. Kretschmer, J. Gouw, J. Rogalski, and N. Scott for discussions and technical assistance. We also thank D. Oliveira for the collection of mouse samples. This work was supported by an NSERC fellowship to JG, CIHR open operating grants to JWK and LJF, and a Burroughs Wellcome Fund Scholar Award in Molecular Pathogenic Mycology (JWK).
JMHG, LJF, and JWK conceived of the study and participated in its design. JMHG carried out the quantitative proteomic sample preparation, data analysis, and interpretation, the targeted proteomic sample preparation, data analysis, and interpretation, validation studies, and drafted the manuscript. DC performed the GO enrichment analyses. MC performed the macrophage assays. NS provided technical assistance for the quantitative proteomic analysis and data processing. JWK assisted in drafting of the manuscript. All authors read and approved the final manuscript.
Lum G, Min XJ. FunSecKB: the Fungal Secretome KnowledgeBase. Database - the Journal of Biological Databases and Curation. 2011;2011: doi:10.1093/database/bar001.


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Supercenters, Markets, Warehouse Clubs & C-Stores - California | United States - Info about discounts, promotions, great deals, warehouse sales, big sales, clearance sales and monster sales in 2019.
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industry_corpus2: Hospitality & Catering


text:
GIFT (Gamete Intra Fallopian Transfer) is a modification of the IVF/ET approach, for which, at least one fallopian tube should be patent and good sperm quality (sperm count and motility) is required.
GIFT is indicated mainly for unexplained infertility and mild endometriosis.
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industry_corpus2: Health & Medicine


text:
Meanwhile, a potential new ethics problem for Rangel is sitting in the garage at the Rayburn House Office Building.
That's where the Ways and Means Committee chairman has kept his 1972 silver Mercedes-Benz since 2003, even after the car stopped working and was no longer registered, said George Dalley, his chief of staff.
House rules prohibit the long-term storage of vehicles in the garage.
Rangel began parking there after he and his wife sold their home on Colorado Avenue NW, Dalley said. After a while, the car wouldn't start. A staffer typically drives Rangel to and from his son's home in Clinton, Md., where he stays while in Washington.
"It was always a question of, 'We need to deal with this,' " Dalley said. "There was always a mixture of inconvenience and procrastination more than there was a feeling of hurry because we were violating the storage rules."
Rangel was not notified that he was in violation, Dalley said. The New York Post first reported the problem yesterday. Now Rangel wants to repair the car and register it in New York, Dalley said.
The staffer said he is also trying to calculate the value of Rangel's use of the parking space over the years, which all lawmakers must declare to the Internal Revenue Service as imputed income.
Rangel, 78, has already requested that the House ethics committee look into inaccuracies in his financial filings and tax returns, his rental of several New York apartments at below-market rates, and his fundraising for an academic center named for him.
He has resisted demands that he temporarily relinquish his chairmanship. Yesterday, Democrats blocked a resolution from House Minority Leader John A. Boehner (R-Ohio) that would force the ethics panel to begin investigating within 10 days and remove Rangel as Ways and Means chairman until the work is done.

industry_corpus2: Politics & Administration


text:
Last month, my distraught daughter entered her grandmother’s ICU room in a Catholic hospital, searching for a place to hang a sentimental picture of sunflowers.
When my wife suggested she use the lone nail protruding from the wall above my mother-in-law’s bed, the nurse blurted out a surprising question: “Where’s Jesus?” She meant the crucifix commonly found in Catholic institutions.
In answer to her perplexing question, my wife tactfully suggested that she search for Jesus in one of the dresser drawers where my father-in-law would likely place any Catholic symbol that offended his very Baptist sensibilities.
Her actions seemed a bit funny at the time, but given a few days’ hindsight, the whole question of “Where’s my Jesus?” makes sense to me now.
It started making sense during the first hour of my current 60-day deployment to a local Air Force base when, in three separate incidents, I misplaced my wallet, my military ID and my laptop. My consecutive search for each loss pushed my blood pressure higher and pegged my anxiety level to the max.
My successive losses left me feeling discombobulated and wondering, along with the nurse, if I was beginning to lose my Jesus.
But the truth is, my consternation came from much more than the misplacement of worldly possessions. In addition to my mother-in-law’s catastrophic stroke and my deployment, I’d just sent two adult children packing under the ultimatum given by nearly every parent to nonworking adult children: work or school or out.
With all of this, I felt myself falling behind in my deadlines for my creative writing degree when suddenly my elderly but very active stepfather died.
Where did I last see Jesus?
Fortunately, I can report he’s not missing. I saw Jesus in the eyes of the newly baptized family in my church last week. I also felt him jogging beside me last month down a rainy stretch of an Oregon beach. And in the hospital, I saw Jesus in the eyes of my father-in-law as he wiped the brow of his wife of 56 years.
My Jesus had been with me all the time. I hadn’t lost him. He wasn’t stowed away in a drawer. He was with me.
I don’t know what you call the strength that sustains you in your moment of greatest need, but I call that strength Jesus.
You might have another word for it. Perhaps you call it love or family or Spirit Guide. But whatever you call it, I think we can borrow a page from my Baptist theology, which tells me that once you get your Jesus, you can’t lose him.

industry_corpus2: Others


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Increasing your Vitamin D intake can drastically reduce your risk of at least 11 different cancers, autoimmune diseases, anemia, the flu, upper respiratory infections. It can prevent acne, asthma, autism, IBS, the list goes on and on. Our vitamin D levels seem at least partially responsible for almost every aspect of our immunity health, and, it bears repeating, most of us are dangerously deficient!
Anyone else have any personal experience with Vitamin D supplementation? I am somewhat new to this absolutely fascinating revelation and would love to learn more about it.
Feedback needed! Whole Foods Market is looking to offer us Buggy Bootcampers an exclusive series of workshops on everything from Healthy Eating to DIY Beauty. They promise to provide treats and valuable knowledge from their team of experts! They are even open to designing a workshop based on our interests/suggestions. Before we commit to them, please reply (only to me) whether getting to the store on High Street Ken would be most convenient to you straight after class, or in the evening (when a husband or sitter can take over)? Secondly, if we organized a class to take place close to the store in Kensington Gardens for this occasion would this be convenient for everyone? Please email me back and let me know your honest thoughts.
Sports Monday: if you aren’t finding yourself humbled by the performances in the World Cup, Tour de France, or Wimbledon, then I rest my case. Anyone who is finding themselves thoroughly inspired by these world class athletes can join me tomorrow as we reach the top of our game right here in Hyde Park!
Tabata Tuesday! Ramp up the intensity as we alternate between toning and cardio intervals. 20 seconds on, 10 seconds off for a burn that’ll earn you more than a few extra calories at dinner time.
Thursday: The Feel-Good Workout! This workout is designed to alleviate all those pesky aches and pains we experience as mothers of little ones. Strengthen weaknesses and correct muscle imbalances with unique toning exercises with a backdrop of heart pumping cardio to rejuvenate your mind, body and soul.
Looking forward to seeing you and your little ones this week!


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Olga Raffault 'Les Picasses' Chinon 2014 (750ML) - PJ Wine, Inc.
"...made from fifty-plus year-old vines in the heart of this excellent vineyard, is outstanding in 2014. This is always a cuvée built for the cellar and the 2014 vintage is no exception. The bouquet is pure and promising, offering up scents of red and dark berries, cigar smoke, a complex base of soil tones, a touch of tree bark, a hint of meatiness and a topnote of menthol. On the palate the wine is deep, full-bodied and very transparent in personality, with a rock solid core of fruit, ripe, chewy tannins, good focus and grip and a long, nascently complex and beautifully soil-driven finish. This is outstanding juice in the making! 2024-2065."

industry_corpus2: Agriculture & Fisheries


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New York City, NY – Marcum Search LLC, the executive recruiting affiliate of accounting and advisory firm Marcum LLP, has appointed Steven Goldmintz to head a new Media and Telecommunications Search Division at the Firm. Mr. Goldmintz joins Marcum Search as a Vice President and will be based in the Firm’s New York City office.
Mr. Goldmintz is a recruiting specialist in the media and telecommunications industry. He comes to Marcum from a leading executive search firm where he was Managing Director of recruiting services for this industry group. Previously, he spent 15 years as an executive with a cable industry supplier and has also been a consultant on pay-per-view projects for numerous studios, as well as for ISPs and network start-ups. He began his career in the sales and marketing departments of TelePrompTer Cable TV, then the nation’s largest multiple system operator (MSO).
Mr. Goldmintz has served as an Executive Officer of the New York Chapter of the Cable & Telecommunications Association for Marketing (CTAM) and is the only executive recruiter to have been honored with CTAM's National TAMI Award, which recognizes exceptional volunteer service. His other professional affiliations include CTHRA, NAMIC, SCTE, CTAM National, WICT, MFM (Media Financial Managers).
“Steve Goldmintz’s dedication to the cable and telecomm industry, with his deep work experience and involvement in the industry’s professional associations, is validation of the excellence that he brings to Marcum Search. His expertise is an important new complement to a recruiting team with resources in staffing for the full breadth of professional services industries,” said Richard Paris, Marcum Search CEO.
Mr. Goldmintz holds a BA in Mass Communications from Queens College. He is a resident of Long Island.
With offices in New York, Florida, California and Pennsylvania, Marcum Search has recruiting practices in numerous sectors including public accounting, finance, tax, administration, human resources, technology and others.
Mr. Goldmintz holds a BA in Mass Communications from Queens College. He is a resident of Long Island. He can be reached at Marcum Search at 212-485-5693 or steve.goldmintz@marcumsearch.com.

industry_corpus2: Computing & Telecommunications


text: Refuse and recycling pickup will be on Saturday Nov 26th. The schedule is different this week to accommodate the Thanksgiving holiday. As a courtesy to others, please refrain from placing your collection carts to the curb any earlier than Noon the day before collection.


text: Website Overview: The server, which itself is located in Cambridge; United States, has an IP address 72.52.4.90. Overall there are 2 off-site links on the homepage of the website. Expiration of tenderuslug.ru will occur on May 31st in 2017. Alexa Global rank for tenderuslug.ru is 977528. Domain was probably registered on May 31st in 2016. This domain's WHOIS records was updated on April 24th in 2017. The past 3 months resulted in Alexa position shift by -139716 for tenderuslug.ru.


text: Websites are great way to start but they can't say everything. If you've got questions or ideas and want to talk possibilities, send an email or use the form below to set up a call. We love a challenge and are open to unique projects.


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It's 1982 and a brand new magic video game cabinet has arrived at the local amusement arcade. somehow the machine is stuck in sound debug mode!?
Pocket arcade synthesizer and sequencer with parameter locks and punch-in effects.

industry_corpus2: Gaming


text:
Major applications now exist in the housing sector, key worker accommodation, student accommodation, schools, hotels, health care and MoD/MoJ accommodation, as well as specialist general applications such as infill walls and framing in commercial and industrial buildings. Through its `Architectural Engineering' research group (based jointly within the Department of Mechanical Engineering and Mathematical Sciences and the School of Architecture) the University is a strategic research partner of Tata Steel Europe. A large amount of the underpinning research has been undertaken using direct industrial funding. Through this, the University has received approximately £1.25 million of support and hosts a Tata sponsored chair held by Professor Ogden.
Early studies comprised theoretical work to predict the competitiveness and supply chain implications of light steel as a mainstream construction material, and identified the technical, structural and building physics research necessary to optimise performance and achieve efficient compliant solutions. This resulted in the generation of widely published and industry supported design data for light steel in the areas of building physics, durability and construction (led by Oxford Brookes University) and structural compliance (led by SCI with Oxford Brookes). This world leading information provided a basis for system developers to begin to offer products into commercial markets on a large scale e.g. Metsec's Metframe and British Steel's `Surebuild' system.
Subsequently, a major proposal for an international multi-partner project focusing on light steel construction, known as MegaProject 5: Steel in the Urban Environment was supported by the EU. Partner countries included UK, Finland, Sweden, Italy, Germany and France. Ogden led the overall portfolio of activities initially for housing and latterly also for the multi-occupancy (apartment) sector. These activities resulted in demonstration projects across the partner countries and the then largest light steel framed building in Europe being constructed at Oxford Brookes University (a three storey house and student hall of residence) together with a number of related full scale test buildings.
The large scale demonstration project at Brookes underwent exhaustive testing and monitoring over a three year period. The building played an essential role in establishing grounded performance data. This was particularly important in areas such as building physics performance (thermal and acoustic), where systems details are critical; and durability and corrosion resistance, which previously had been an area of systematic and premature failure due to poor detailing and lack of technical understanding. Complementary to this, full scale test buildings constructed in laboratories provided actual structural performance data which then allowed engineers to design beyond conservative structural code predictions. The work led to a sustained programme of research and development from 1997, which is still on-going in the context of advanced applications (e.g. high rise modular in excess of 7 stories). The research resulted in further technical outputs, (5-10), and further research grants (11).
Subsequent to EU MegaProject 5 and in parallel with its portfolio of funded research for Tata Steel, Oxford Brookes has provided applied research and development services to downstream industry in the area of light steel framing. It supported major investments from companies including Terrapin, Barratt, and Corus Living solutions, as well as overseas companies such as Posco in South Korea. It maintains a dedicated and intensively used structures and building physics laboratory. Examples of applied activities also include Ogden's buildings concept around which Unite Modular Solutions commissioned a major production facility in Gloucestershire able to produce three room modules per hour/6000 modules pa.
1. Industry Funding: Circa £1.2m in the period 2002-2013. Oxford Brookes Umbrella Research Agreement with Tata Steel. PI Prof. R G Ogden. 9.9.2002.
2. Grant: MegaProject 5: Steel in the Urban Environment. European Coal and Steel Community (ECSC). Contact ref: 7215-CA/808. Contract period 1.7.1994-31.12.1997. Project cost 837k Euro.
3. Demonstration Project: Light steel framed student accommodation building. Oxford Brookes University (John Garne Way) .1997. Lead industrial partners: British Steel (`Surebuild') and Taywood Homes.
Submitted to RAE2001, Oxford Brookes University, UoA33-Built Environment, RA2, RG Ogden, Output 2.
4. Grant: MegaProject 5: Valorisation Phase (Dissemination) Steel in Housing. European Coal and Steel Community (ECSC). Contact ref: 7215-PP/043. Contract period 1.7.1999-30.6.2001. Project cost 280K Euro.
8. Refereed journal article: Developments in prefabricated systems in light steel and modular construction. The Structural Engineer, Volume 83, No 6, Lawson, M., Ogden, R., Pedreschi, R., Grubb, J. and Popo-Ola, S. 2005.
Submitted to RAE2008, Oxford Brookes University, UoA31-Town and Country Planning, RA2, RG Ogden, Output 2.
Submitted to RAE2008, Oxford Brookes University, UoA31-Town and Country Planning, RA2, RG Ogden, Output 4.
Submitted to REF2014, Oxford Brookes University, UoA15-General Engineering, REF2, RG Ogden, Output identifier 7812.
11. Grant: Demonstration of Modular Steel Construction in the Renovation of Multi-Storey Residential Buildings. European Coal and Steel Community (ECSC). Contact ref: 7215-PP/010. Contract period 1.7.1997-31.12.2000. Project cost 1950K Euro.
As lead academic partner, the world leading information generated by Ogden and his team at Oxford Brookes University has played a central role in developing and proving the merits of light steel technology. Various examples of publications and demonstration activities arising from the research are cited in section 3, however these represent a small proportion of the total industry accepted outputs. Projects such as the Oxford Brookes European MegaProject 5 demonstration building played not only a key role in developing the technology but also in creating awareness and understanding. The building received over 2000 visitors and gained extensive press coverage (circa 40 articles written between April 1997 and March 1998).
Companies have been prepared to commit to multi-million pound investment programmes to deliver products at commercial volumes based on the demonstrated technologies (e.g: Ayrshire, BW Industries, Fusion, Kingspan, Knauf, Lafarge, British Gypsum. Metsec).
Specifiers have gained confidence in the performance of light steel framing such that they are willing to use it in preference to longer established but often less efficient alternatives.
Building control agencies (such as local authorities, Lantac, NHBC etc.) have accepted that systems are free of unacceptable or abnormal levels of risk. Buildings based on the design practices that have been developed are considered equally by mortgage and funding companies to those constructed using conventional technologies such as masonry and timber.
The scale of impact of the research can in part be gauged by the major contribution that the market makes to the steel production sectors where the annual tonnage is over 1% of total UK output.
The market share of light steel framing is currently 17% overall and gaining on timber frame, currently 56% (a). In the permanent modular off-site buildings market, light steel framing has essentially taken over from timber to the extent that few timber framed modular companies remain.
28% of the total UK offsite construction market is attributed to permanent light steel modular buildings of the type addressed by the research, a further 9% to relocatable buildings (approximately 75% of which rely on light steel framing). The increasing efficiency of modular construction has meant that it has grown from a negligible amount in 2000 to a point where it is currently estimated to account for 2% of the total construction market (b).
Based on annual sector surveys by the independent market research company `Construction Markets' (c), the peak light steel framing market in the period 2008-2012 was 66K tonnes pa. Taking an average galvanised strip steel cost of £600 per tonne this equates to a materials market of £39.5 million pa. The contribution of this to the turnover of supply chain companies (assuming normal manufacturing overheads of 1.75 and margins of 12.5%) is circa. £78 million pa. to the UK economy.
This use of domestically produced product is actively reducing timber imports, as conventional and pre-existing technologies require softwood sections not available in the UK. The effect on the UK trade balance is therefore significant.
It is important to recognise that this impact has been achieved in a recessionary period where the total construction market has been reduced by approximately 50% and where conventional costs are at their most competitive.
Improved speed of construction with rapid reliable construction programmes.
Higher levels of automation reduce costs and reliance on specialist site trades.
Improved quality with low levels of defect remediation required post construction and reliable in service performance.
Improved environmental performance including low waste, good insulation and high levels of airtightness.
These benefits of light steel construction developed in the UK are also reflected in the international development of the technology. In part this is due to the improved resilience and robustness against earthquake and wind loading. Early adopters therefore include New Zealand, Australia, Japan, USA, Canada and South Africa (h).
The knowledge gained in relation to light steel within the team has also formed the foundations for commercial innovation, such as the creation of high reflectivity liner sheets which have been developed for built-up cladding systems. Launched in late 2012, these have sold 1000 tonnes by May 2013 value £600K.
a. Market research: www.amaresearch.co.uk/prefabricated_frame_panel_12s.html, as available 29/05/2013.
b. Report: Offsite Production in the UK construction Industry. A Brief Overview. Taylor, S. Construction Engineering Specialist Team: Health and Safety Executive, 2009.
c. Market research: Annual Independent statistics (2008-2012) compiled by `Construction Markets Ltd'. Collins, D.J., 2012.
d. Refereed journal article: Benefit evaluation for off-site production in Construction. Construction Management and Economics, Volume 24, No 2, 121-130. DOI: 10.1080/01446190500184444. Blismas, N., Pasquire, C. and Gibb, A., 2006.
e. Report: Offsite Housing Review. Report funded by the Department of Business, Innovation & Skills and the Department for Communities & Local Government through the Construction Industry Council (CIC), with Oxford Brookes University and the University of Cambridge. Whitehouse, N. and Miles, J., 2013.
g. Report: Value and benefit assessment of light steel framing in housing. Rogan, AL and Lawson, RM, Steel Construction Institute, 1998.
h. Journal article: Light steel framing at Steelfuture conference. Steel Construction, Volume 37, No 2, Barnard, J., 2013.
i. Corroborating contact 1. Director of Strategic Marketing (responsible for all European business sectors), Tata Steel.
j. Corroborating contact 2. Former Product and Market Development Manager (responsible for UK and EU market development within British Steel Strip Products).
k. Corroborating contact 3. Former construction policy specialist.

industry_corpus2: Real Estate & Construction


text:
L x W x D: 15" x 15" x 15"
Compare to Uline S-4369 and Save!
Our 15 x 15 x 15-13-11-9-7 Multi-Depth boxes offer the benefits of having many box sizes in your inventory to suit your various packaging needs and save warehouse space. Box sides are scored to multiple depths to allow height adjustment for a perfect fit. Our 15'' x 15'' Multi-Depth style boxes adjust to the heights of 15", 13", 11", 9" and 7". These 15" x 15" shipping boxes meet requirements for 200#-C/ECT-32-C corrugated. Great for shipping or storing medium size items.

industry_corpus2: Manufacturing


text:
June 1906 photograph of a young woman identified on the reverse as Rena Frances Johnson. The photograph lacks a photographer's imprint.
Rena Frances Johnson was born 10 June 1888 at Corinth, Maine, daughter of Albert and Harriet Ellen (Jaquith) Johnson, who were born at Corinth, Maine, and Albion, Maine, respectively.
Rena's paternal grandparents were, I believe, John and Frances (Peterson) Johnson. Her maternal grandparents were Obed C. and Betsey M. (Stevens) Jaquith.
On 1 May 1908 at Corinth, Maine, Rena married John Forrest Tate, son of William and Irene W. (Gerry) Tate. He was born 25 December 1875 at Charleston, Maine, which abuts Corinth.
They lived at Corinth for a while and eventually moved to Portland, Maine, where John worked as a house carpenter. They had at least three children. John died in Florida in 1926.
On 29 October 1929 at Auburn, Maine, Rena married Floyd Monroe Barrows, son of James B. and Jane (Davis) Barrows. He was born at Oakfield, Maine, on 11 March 1883. Floyd had earlier been married to teacher Cora Louise Shields, daughter of Calvin and Emily V. (Brown) Shields. She died in 1917.
The 1930 Census found Floyd and Rena at Auburn, Maine, where Floyd was working as a farmer, and Rena was at an Academy. They were still at Auburn by the time of the 1940 Census. Floyd's occupation was still listed as farmer, but no occupation was given for Rena.
Rena died at Mount Desert, Maine, in 1974. Floyd had died in 1960.
If you have corrections to the information above or information on the Johnson, Tate or Barrows families, please leave a comment or contact me directly. Thanks!


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Jason is available for photography and moving image projects as well as other potential adventures worldwide. Jason is also proficient and interested in work in creative strategies, social media, event planning and promotion, location scouting, and art direction, as well as music and voice over production.
Jason can be reached via email at jason.walsmith@gmail.com or by telephone at 515-664-3333. You can also use the form below to inquire about rates and availability, or just to say hello.

industry_corpus2: Film & Entertainment


text:
If there are two input ports in a router waiting to use a particular output port, then an arbitration mechanism is used to select which input port is to be served. The arbitration mechanism can include a priority scheme. There is no priority flag available within the header of a SpaceWire packet to specify its priority level. The SpaceWire header only contains address information, so packet priority must be associated with a logical address (or with the input port number). In the routing tables logical addresses may be assigned high or low priority. High priority logical addresses have preferential access to an output port when arbitration takes place. A logical address that has been assigned high priority, acts as a high priority channel across the network from many possible sources to the one destination. If high and low priority access to a particular destination is required then two logical addresses are required for a particular destination, one assigned high priority and the other low priority. A source can then decide which logical address to use when sending a packet to a destination, depending on the required priority of the packet. There is a compromise between the number of destinations that can be addressed and the number of priority levels. With two priority levels it is possible to have, for example, 128 low priority destinations and 96 high priority destinations within the 224 logical addresses available. Priority is a means of providing quality of service control.
It is possible to ensure real-time, deterministic delivery of commands (packets) using priority addresses provided that there are no possible clashes between routes through the network operating priority addressing. This can be achieved, for example, if there is only one node sending out priority commands. If deterministic delivery is required there must also be a limit to the maximum packet size used on the network.
Alternatively nodes can take turns to transmit their high priority packets possibly using time-codes to determine which node transmits next.


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Kurumbapalayam, Coimbatore, Tamil Nadu – 641107.
SNS College of Engineering was established in the year 2007 with a vision of being in the forefront of Technical education and Management studies. The motto of our Institution is to impart quality education with an international exposure leading to knowledge and develop multiple skills among the students community. With a state-of-the-art infrastructure, highly skilled & dedicated faculty members and outstanding placement & training cells with successful proven track records, SNSCE keenly concentrates on continual knowledge enrichment that moulds the students to achieve excellence in all their endeavors. SNSCE strives to develop competent and committed professionals driven by values, who aim to take over the corporate world as its crusaders. SNSCE has tie-ups and MoUs with multi-national corporate companies to facilitate industrial and corporate exposure for students and faculty. The college is affiliated to Anna University, Chennai and is approved by AICTE, New Delhi.

industry_corpus2: Subject Education


text:
NORTH CHARLESTON, S.C.: South Carolina authorities say a 44-year-old woman angry at a man for returning home without beer on Christmas beat and stabbed him with a ceramic squirrel.
The Charleston County Sheriff�s office says in a report that deputies found a man covered with blood when they arrived at Helen Williams� North Charleston home early Wednesday. She told investigators the man fell and cut himself, but couldn�t explain why her hands and clothes were also bloody.
Williams was in jail Friday and charged with criminal domestic violence. It wasn�t known if she had a lawyer.

industry_corpus2: Law & Justice


text:
A moderate westerly breeze tempered conditions for the 71 starters in the the Intersport Degrandi 21.1 km handicap along the Barwon River paths. The runners certainly appreciated the tail wind in the final two kilometres. It was Peter McDonough who forged to the lead inside the last 600 metres to go on to win by 90 metres from Mark Humphrey with Jeff Walker a further 20 metres away third. McDonough, who has struggled with injury since recording two victories in 2013, put the writing on the wall with a placing at Anglesea last Sunday. Sara Coulter was the fastest female by over 12 minutes whilst Zac Boothroyd recorded his first ever fastest in the male division.
Sean Thompson was congratulated on reaching his 100 run milestone.
P.McDonough, 104:07; M. Humphrey, 95:28; J. Walker, 105:03; C. Bridgland, 122:44; E. Sims, 105:12; M. Maiden, 92:50; B. O’Neill, 82:36; S. Coulter, 88:42; A. Ruggero, 82:39; S. Van Rooyen, 84:11.

industry_corpus2: Sports