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- Research
- Open Access
Histoplasma capsulatum proteome response to decreased iron availability
© Winters et al; licensee BioMed Central Ltd. 2008
- Received: 10 September 2008
- Accepted: 24 December 2008
- Published: 24 December 2008
A fundamental pathogenic feature of the fungus Histoplasma capsulatum is its ability to evade innate and adaptive immune defenses. Once ingested by macrophages the organism is faced with several hostile environmental conditions including iron limitation. H. capsulatum can establish a persistent state within the macrophage. A gap in knowledge exists because the identities and number of proteins regulated by the organism under host conditions has yet to be defined. Lack of such knowledge is an important problem because until these proteins are identified it is unlikely that they can be targeted as new and innovative treatment for histoplasmosis.
To investigate the proteomic response by H. capsulatum to decreasing iron availability we have created H. capsulatum protein/genomic databases compatible with current mass spectrometric (MS) search engines. Databases were assembled from the H. capsulatum G217B strain genome using gene prediction programs and expressed sequence tag (EST) libraries. Searching these databases with MS data generated from two dimensional (2D) in-gel digestions of proteins resulted in over 50% more proteins identified compared to searching the publicly available fungal databases alone. Using 2D gel electrophoresis combined with statistical analysis we discovered 42 H. capsulatum proteins whose abundance was significantly modulated when iron concentrations were lowered. Altered proteins were identified by mass spectrometry and database searching to be involved in glycolysis, the tricarboxylic acid cycle, lysine metabolism, protein synthesis, and one protein sequence whose function was unknown.
We have created a bioinformatics platform for H. capsulatum and demonstrated the utility of a proteomic approach by identifying a shift in metabolism the organism utilizes to cope with the hostile conditions provided by the host. We have shown that enzyme transcripts regulated by other fungal pathogens in response to lowering iron availability are also regulated in H. capsulatum at the protein level. We also identified H. capsulatum proteins sensitive to iron level reductions which have yet to be connected to iron availability in other pathogens. These data also indicate the complexity of the response by H. capsulatum to nutritional deprivation. Finally, we demonstrate the importance of a strain specific gene/protein database for H. capsulatum proteomic analysis.
- Heat Shock Protein
- Protein Spot
- Malate Dehydrogenase
- Inductively Couple Plasma Mass Spectrometer
- Mass Spectrometer Data
Histoplasma capsulatum is a dimorphic fungus and the etiological agent of histoplasmosis. The fungus is endemic to the Midwestern and southeastern United States. H. capsulatum can be a life-threatening infection for individuals suffering from immune defects associated with AIDS and for those receiving immunosuppressive pharmaceuticals to combat malignancy, graft rejection, and autoimmunity. More recently, antagonists to tumor necrosis factor-α have been identified as a risk factor for the development of disseminated histoplasmosis [1–5].
H. capsulatum survives in the mammalian host by evading both the innate and adaptive immune responses. H. capsulatum is confronted by several phagocytic cell populations including immature dendritic cells, neutrophils, and macrophages. Among them, the last is the only professional phagocytic cell population in which H. capsulatum can replicate freely [6]. Intracellularly, H. capsulatum must confront and adapt to phagolysosomal fusion, nitrosative stresses, low nutrient availability, and low pH [7–12].
Two-dimensional electrophoresis in combination with mass spectrometry has become a powerful tool for studying the proteome of a number of intracellular pathogens [13–19]. There have been limited microarray and proteomic analyses of H. capsulatum [20–22]. For fungi such as H. capsulatum, such information is nascent. Therefore we have utilized the publicly available H. capsulatum G217B genome and transcripts developed for microarray analysis to construct protein/genomic databases compatible with mass spectrometry (MS) data search engines. We have tested the utility of these databases and proteomics for H. capsulatum by studying the response under iron-limiting conditions.
Iron is a major nutrient for growth of H. capsulatum both in vitro and in vivo [23–25]. Sequestration of iron by interferon gamma (IFN-γ) activated macrophages acts as a host defense mechanism against H. capsulatum [26]. The influence of iron availability on metabolic processes in H. capsulatum has only begun to be addressed. Recently a connection between iron availability and lipid metabolism was shown in H. capsulatum as iron-related alterations influenced the content of triacylglycerol and free fatty acids [27]. We have utilized 2D gel electrophoresis, statistical analysis, MS, and bioinformatics to identify H. capsulatum proteins sensitive to lowering iron availability. Using this platform, we discovered 42 H. capsulatum proteins whose abundance was altered when grown in the presence of iron scavenger apo-transferrin. Proteins found to be sensitive to decreasing iron levels suggest a requirement for H. capsulatum to mediate specific metabolic functions in order to cope with changes in the environment the organism most likely encounters in the host.
Results and Discussion
Proteomic platform development
We searched matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (MS) and nanoflow liquid chromatography (LC)-MS/MS data obtained from 2D in-gel digests of protein spots labeled in figure 1 against all the MASCOT searchable publicly available eukaryotic databases. Protein spots were selected based on intensity in order to test the quality of the different gene/protein databases to generate the most protein identifications. Following download and MASCOT modifications we searched the G186 database available at the Broad Institutes Fungal Genome Initiative. http://www.broad.mit.edu/annotation/fgi/. Searching the public available databases and Broad yielded identifications for 34 out of 49 individual protein in-gel digests. In order to increase our protein identifications, a gene/protein database specific for the G217B H. capsulatum strain and compatible with MS search engines was constructed using a combination of different gene finding programs.
Database 1, consisting of ~9348 sequences, was constructed using Fgenesh and parameters setup by Softberry http://www.softberry.com/berry.phtml. Fgenesh finds genes based on intrinsic characteristics of coding sequences such as codon usage and consensus splice sites [28]. Database 2 was assembled using the publicly available EST libraries of both mycelial and yeast phases, and assembled into full gene sequences with the aid of the CAP3 genome assembly program [29] yielding a database containing 6,233 gene sequences. Database 3 was originally assembled by Washington University St. Louis Genome Sequencing Center for microarray analysis using a combination of different gene finding programs which include Fgenesh, EAnnot, GeneWise, and SNAP [28, 30–32]. Database 3 contains a total of 14,506 predicted gene sequences.
List of proteins identified from the 2D gel shown in figure 1 following in-gel tryptic digestion, MALDI-MS analysis, and database searching.
Spot #
# Peptides Matched/#Searched
% Protein Coverage
Accession #
MASCOT Score (p < .05)
Spot 1*
predicted protein [Ajellomyces capsulatus NAm1].
Spot 2
aconitate hydratase, mitochondrial precursor [Ajellomyces capsulatus NAm1].
Spot 3
heat shock 70 kDa protein C precursor [Ajellomyces capsulatus NAm1].
Spot 4
heat shock protein 70 [Paracoccidioides brasiliensis].
Spot 5 (A10)
70 kDa heat shock protein [Paracoccidioides brasiliensis].
Spot 6*
protein disulfide-isomerase precursor [Ajellomyces capsulatus NAm1].
Spot 7
heat shock 70 kDa protein [Ajellomyces capsulatus NAm1].
Spot 8
heat shock protein 60, mitochondrial precursor [Ajellomyces capsulatus NAm1].
Spot 9
ATP synthase beta chain, mitochondrial precursor [Ajellomyces capsulatus NAm1].
Spot 10 (A21)
enolase [Paracoccidioides brasiliensis].
Spot 11
ubiquinol-cytochrome C reductase complex core protein 2, putative [Aspergillus fumigatus Af293].
Spot 12
dihydrolipoamide dehydrogenase [Ajellomyces capsulatus NAm1].
Spot 13*
fumarate hydratase class II [Aspergillus terreus NIH2624].
Spot 14*(A29)
saccharopine dehydrogenase [Ajellomyces capsulatus NAm1].
Spot 15
elongation factor Tu, mitochondrial precursor [Aspergillus terreus NIH2624].
Spot 16
pyruvate dehydrogenase E1 component alpha subunit, mitochondrial precursor [Ajellomyces capsulatus NAm1].
Spot 17 (A38)
malate dehydrogenase [Ajellomyces capsulatus NAm1].
Spot 18
glyceraldehyde-3-phosphate dehydrogenase [Ajellomyces capsulatus].
Spot 19
ketol-acid reductoisomerase, mitochondrial precursor [Ajellomyces capsulatus NAm1].
Spot 20
mannitol-1-phosphate dehydrogenase [Paracoccidioides brasiliensis].
Spot 21
conserved hypothetical protein [Ajellomyces capsulatus NAm1].
Spot 22
peptidyl-prolyl cis-trans isomerase [Ajellomyces capsulatus NAm1].
Spot 23
peptidyl-prolyl cis-trans isomerase [Aspergillus fumigatus Af293].
Spot 24*
fructose 1,6-biphosphate aldolase 1 [Paracoccidioides brasiliensis].
Spot 25*
RACK1-like protein [Paracoccidioides brasiliensis].
Spot 26*
cytochrome c peroxidase Ccp1 [Aspergillus fumigatus Af293].
Spot 27*
protein disulfide-isomerase precursor [Ajellomyces capsulatus NAm1].
Spot 28
gene encoding Histoplasma capsulatum predicted protein (2481 nt)
Spot 29*
mRNA binding post-transcriptional regulator (Csx1) [Aspergillus fumigatus Af293].
Spot 30*
gene encoding Histoplasma capsulatum predicted protein (2328 nt)
Spot 31*
hypothetical protein RUMOBE_03015 [Ruminococcus obeum ATCC 29174]
Spot 32
Spot 33
Hsp90 binding co-chaperone (Sba1) [Aspergillus fumigatus Af293].
Spot 34
thiol-specific antioxidant [Ajellomyces capsulatus NAm1].
Spot 35*
triosephosphate isomerase [Aspergillus clavatus NRRL 1].
Spot 36
30 kDa heat shock protein [Ajellomyces capsulatus NAm1].
Spot 37
superoxide dismutase, mitochondrial precursor [Ajellomyces capsulatus NAm1].
Spot 38
ATP synthase D chain, mitochondrial [Ajellomyces capsulatus NAm1].
Spot 39
tropomyosin, putative [Neosartorya fischeri NRRL 181].
Spot 40*
protein of unknown function DUF181 [Methylobacterium radiotolerans JCM 2831].
Spot 41
peptidyl-prolyl cis-trans isomerase (cyclophilin) [Ajellomyces capsulatus NAm1].
Spot 42
putative cytochrome c oxidase subunit VIa [Paracoccidioides brasiliensis].
Spot 43
woronin body major protein [Ajellomyces capsulatus NAm1].
Spot 44 (A5)
HCAG_04147 (Hypothetical Protein)
Spot 45*(A42)
asparaginyl-tRNA synthetase [Flavobacterium sp. MED217].
Spot 46
ATPase associated with various cellular activities AAA_3 [Shewanella woodyi ATCC 51908]
Spot 47*
cystathionine beta-lyase [Rhizobium etli CFN 42].
Spot 48
heat shock protein 70 [Paracoccidioides brasiliensis].
Spot 49
hypothetical protein NCU07591 [Neurospora crassa OR74A].
The protein identifications (BLAST IDs) listed in table 1 are the best homology and functional matches following a BLAST search of the MS matched protein sequence in our custom databases against the NCBInr sequence database. If a protein sequence matched with a hypothetical sequence in the BLAST search, the next best protein BLAST sequence match with annotated functionality was listed. Forty percent of proteins detected are involved in energy or metabolism; 30% belonged to the cell fate and rescue functional cluster. The most intensely SYPRO stained protein bands were heat shock proteins (HSP)s. In addition, MS data from spots 32 and 44 (Figure 1) matched two protein sequences in the Broad 186 strain databases only, neither of which had homology to any other protein sequence following BLAST searching.
Our mass fingerprinting (MFP) analysis was validated using a more sensitive and structurally informative technique, LC-MS/MS analysis. We analyzed 3 digested proteins (Figure 1 spots; 6, 8, and 41) by MFP and LC-MS/MS (table 1) and all 3 matched with their respective protein using both MFP and LC-MS/MS. This included peptide sequence information for 14 fragments from spot 6, six fragments from spot 8, and 3 from spot 41.
Databases were further validated using MS data acquired from in-gel digestion of HSP 60. MS data from in-gel tryptic digested HSP 60 matched exceptionally well with both public and in-house constructed databases. We matched 18 MS generated molecular weights with 18 amino acid sequences to our in-house constructed databases for HSP 60. Using the same MS data to search the publicly available fungal databases using MASCOT, we matched 13 peptides. This also shows the specificity of our databases for H. capsulatum, in that we were able to identify 5 additional peptides unique to H. capsulatum G217B, HSP 60 (data not shown).
Iron depletion and 2D gel analysis
Because iron utilization is crucial for successful H. capsulatum infection and limiting iron to the organism is believed to be a strategy used by the host, we analyzed H. capsulatum's protein response to decreases in iron availability. H. capsulatum requires iron for survival inside the host as chelation of iron inhibits fungal growth within mouse and human macrophages [23, 26]. In mammalian hosts iron is bound to transferrin and lactoferrin or stored in ferritin. It has been proposed that H. capsulatum may acquire iron from transferrin by modulating pH [23] or by secreting siderophores [33]. Transcripts of genes coding for siderephore biosynthesis are upregulated under low iron conditions, and one gene, SID1, is involved in host colonization [34]. Another iron acquisition strategy is through the secretion of ferric reducing compounds [25, 35]. Recently it has been shown that as H. capsulatum has a preference for ferrous iron [27], and a possible mechanism for H. capsulatum iron acquisition is through the ferric reductant activity of a γ-glutamyltransferase [36].
Proteins with statistically significant spot volume changes are circled in white, numbered in Figures 2B and 2D. The 42 proteins circled in white represent fold changes in a range of 1.3–2.8, and p values < .05. Spot volume ratios of all proteins detected and matched using SameSpots were subjected to a global one-way ANOVA statistical test. ANOVA was used to define a subset of individual proteins that exhibited statistically significant abundance changes (normalized volume ratios) as one-way ANOVA analysis is designed to test differences between means in multiple sample groups. Twenty four and 48 hr groups were analyzed separately.
Proteins sensitive to changes in iron availability
Seven H. capsulatum proteins were more abundant while 35 proteins were less abundant following growth in iron-poor (apo-transferrin) medium. A list of these proteins and their corresponding fold changes and p values can be found in the additional file 1. Protein spots identified by MS are labeled A for apo-transferrin in table 1. Protein sequences with homology to enolase (spot A21 Figure 2D), malate dehydrogenase (spot A38 Figure 2D), saccharopine dehydrogenase (spot A29 Figure 2D), HSP 70 (spot A10 Figure 2D), asparaginyl-tRNA synthetase (spot A42 Figure 2D), and an uncharacterized protein with an amino acid sequence unique to H. capsulatum (spot A5 Figure 2B) were among the 35 proteins less abundant following H. capsulatum growth in apo-transferrin medium. Fold changes and p values calculated by the SameSpots software were as follows; enolase (-1.4 fold change/p value = .0193), malate dehydrogenase (-1.4/.02920), saccharopine dehydrogenase (-1.5/.0377), HSP 70 (-1.4/.0227), asparaginyl-tRNA synthetase (-1.4/.0422), and an uncharacterized protein unique to H. capsulatum (-1.4/.0293). This protein was less abundant during the first 24 hr incubation with apo-transferrin and returns to baseline another 24 hr later (Figure 2D, A5).
Enolase and malate dehydrogenase are involved in cellular respiration steps of the tricarboylic acid (TCA) or Krebs cycle and the last step of glycolysis before entering the TCA cycle respectively. Enolase, malate dehydrogenase, and other transcripts involved in the TCA cycle were also less abundant following microarray analysis of C. albicans, C. neoformans, and Escherichia coli under iron depletion conditions [37–39]. Fold changes of 1.56 and 1.87 for enolase and malate dehydrogenase transcripts respectively detected by microarray analysis of C. neoformans [38] under reduced iron availability was similar to those observed in our analysis. Isolating H. capasulatum following ingestion from IFN-γ activated macrophages we found that malate dehydrogenase was also significantly altered in abundance (fold change of 5.6, p = 0.0153) compared to the organism grown under the same conditions in culture (additional file 2). When faced with limitation of iron, yeasts will not only increase their iron uptake and their mobilization of stored iron, but also adjust their metabolism to more efficiently use the iron that is available [40]. The availability of iron in H. capsulatum has also been shown to affect the amount of triacylglycerols implicating a potential role of iron in lipid metabolism[27].
In addition to proteins involved in cellular respiration, we observed HSP 70 less abundant in H. capsulatum when grown in an iron deficient environment. HSP 70 is involved in the biogenesis of iron cluster proteins. Again our results are consistent with what was observed following microarray analysis of transcripts which code these proteins in C. ablicans following iron depletion [37]. HSPs may chaperone Fe-S proteins or may be involved in the transfer or iron centres to recipient proteins thus their productivity may decrease when iron levels are lowered.
We also discovered proteins with sequence homology to saccharopine dehydrogenase and asparaginyl-tRNA synthetase whose abundance was decreased when H. capsulatum's growth medium iron concentrations were reduced. Saccharopine dehydrogenase is an enzyme involved in lysine metabolism while tRNA synthetase enzyme is required for protein synthesis by catalyzing the specific attachment of asparagine to its cognate tRNA. Out of ~1500 proteins profiled in our model we observed ~2.0% reduction in protein abundance when H. capsulatum was incubated with apo-transferrin for 48 hr. Taken together these data could suggest a global reduction in protein synthesis by H. capsulatum when confronted with reductions in iron availability. To the authors knowledge neither asparaginyl-tRNA synthetase or saccharopine dehydrogenase have been connected to any other organism's response to lowering iron levels.
Western blot analysis
Trifluoroacetic acid (TFA) was obtained from Applied Biosystems (Warrington, WA). High performance liquid chromatography (HPLC)-grade acetonitrile, methanol, acetic acid, HPLC-grade water, ammonium bicarbonate, dithiothreitol (DTT), acrylamide, iodoacetamide, urea, and 3-[3-(Cholamidopropyl) dimethylammonio]-1-proanesulfonate (CHAPS) were obtained from Fisher (Fair Lawn, New Jersey). Carrier ampholytes (pH 3–10) and formic acid were purchased from Fluka Chemicals (Milwaukee, WI). SYPRO Ruby fluorescent dye was obtained from Invitrogen Molecular Probes (Carlsbad, CA). α-Cyano-4-hydroxycinnamic acid, bathophenathrolinedisulfonic acid (BPS), and protease inhibitor cocktail for fungal and yeast cells (P8215) was purchased from Sigma Aldrich (St. Louis, MO). Sequence grade trypsin was obtained from Promega (San Luis Obispo, CA). 4-chloro-1-naphthol HRP development was obtained from BioRad (Hercules, CA). HRP conjugate secondary antibodies were purchased from Pierce (Rockford, IL). HSP70 mouse monoclonal antibody was purchased from Sressgen (Victoria, BC Canada). Cyclophilin mouse monoclonal antibody was a gift from Francisco Gomez (University of Cincinnati, Infectious Disease).
Sample preparation
H. capsulatum yeast (strain G217B) were prepared by inoculating 50 ml of medium, previously described [41], with 3 × 106 cells/ml from 3–5 days old slants to give a concentration of 1.5 × 108 cells/ml after 3 days. H. capsulatum yeasts were grown in medium set at pH 7.5 for 48 hr and switched to medium containing 5 μM apo-transferrin or medium free of apo-transferrin. One ml aliquots (~108 cells) were removed following 24 hr and 48 hr incubation with 5 μM apo-transferrin and medium free of apo-transferrin. Samples were then centrifuged at 1500 × g for 10 min. The supernatant was removed and 250 μl of lysis buffer (9 M urea, 2% CHAPS, 1% DTT, and 10 mM fungal and yeast cell protease inhibitor) was added. Cells were disrupted with a Min-Beadbeater (BioSpec Products, Bartlesville, OK) using an equal volume of glass beads for 30 sec and placed on ice for 30 sec. This was repeated 3 times. Following cell disruption, 250 μl of lysis buffer was added and samples were allowed to cool on ice for 10 min. Samples were then centrifuged at 15,000 × g for 30 min. An equal volume of water was added to each sample and further concentrated to 100 μl using an Amicon Ultra Centrifugal Filter Device. The clear supernatant was removed and protein concentrations were determined using the Bradford method.
We confirmed that apotransferrin did reduce iron levels by measuring the amount of iron bound to transferrin using the iron chelator bathophenathrolinedisulfonic acid (BPS) as described previously [42, 43]. One ml of media containing 5 μM apo-transferrin was concentrated using Amicon Ultra Centrifugal Filter Device. To the concentrated transferrin (100 μl), nitric acid was added to give a 3% concentration followed by the addition of 38 mg/ml sodium ascorbate, 1.7 mg/ml BPS, and ammonium acetate (saturated ammonium acetate dilute 1/3) were added to the concentrated transferrin effectively denaturing the protein and reducing all iron to the ferrous form. After 5 min the absorbance was recorded at 535 nm for the concentrated retintate and flow through. Based on measuring the amount of iron bound to apo-transferrin the quantity of iron in the medium prior to addition of apotransferrin was .0008 g/L and following 24 hr in 5 μM apo-transferrin the concentration was reduced by ~83%. At 5 μM apo-transferrin changes in the amount of iron bound to apo-transferrin in the presence or absence of H. capsulatum was not detected.
Mass spectrometry was also used to measure the reduction of iron levels. The inductively coupled plasma mass spectrometer (ICP-MS) based quantification method has been commonly used to determine accurate concentration of metals in complex matrices. Specific iron detection in our samples was performed with an ICP-MS 7500cx (Agilent Technologies, Tokyo, Japan) equipped with an octopole collision/reaction cell system. Two isotopes of iron, 56Fe+ and 57Fe+, were monitored using He as collision gas at 4.0 ml min-1 to reduce the 40Ar16O+ and 40Ar16O1H+ interferences. The RF power was 1450 W and carrier gas flow rate was 1.02 L min-1. Apo-transferrin medium was filtered through a 10 kD membrane filter before analyzed by ICP-MS. Total iron levels were lowered from 57.15 ppb (± .84), to 5.93 (± 1.27) ppb when 5 μM of apo-transferrin was added to the medium, a reduction of ~90% of the total iron. Standard deviations were calculated from 3 biological replicates.
2D-gel electrophoresis and statistical analysis
Fifty μg of protein were mixed with rehydration buffer (9 M urea, 2% CHAPS, 1% dithiothreitol (DTT), and carrier ampholyte (pH 3–10) solution, 20 μl/1 ml). A total of 350 μl of this rehydration solution was loaded onto 24 cm immobilized protein gradient (IPG) strips pH 3–10 (Amersham Biosciences, GE Healthcare, Piscataway, NJ) and incubated (rehydrated) for 12 hr. Isoelectric focusing (IEF) was performed with an IPGphor system (Amersham Biosciences) using a step gradient starting from 0–500 V for 1 hr, 500–1000 V 1 hr, 1000–8000 V 12.5 hr, and 8000V for 2.5 hr. Following IEF, strips were stored at -70°C.
Strips were then equilibrated for 30 min in 15 ml of 6 M urea, 20% glycerol, .05 M Tris, 2% SDS, 75 mg DTT, and a few crystals of bromophenol blue. Strips were then loaded onto a 10% SDS PAGE gel. The second dimension was run at 200 V for 15 min followed by 300 V for 4–5 hr. Gels were next fixed for 15 minutes in 10% methanol/7% acetic acid. Gels were then stained overnight with SYPRO Ruby fluorescent dye and destained for 20 minutes in fixing solution. Gel images were produced using a GE Healthcare Typhoon scanner. Before image analysis, gels were washed with water for 5–10 min.
2D gel spot detection, matching, and spot volume, calculations were performed using SameSpots (Nonlinear Dynamics, Durham, NC) software on three biological replicates, six gels per 24 and 48 hr apo-transferrin treatments and their respective controls. Spot volumes for all protein spot matches within each analysis were calculated and normalized to the total spot volume yielding a spot volume ratio. This ratio enabled the calculation of average abundance changes across all 3 replicates within each test group and the application of ANOVA statistical test. Individual spots were ranked by p-value from a one way global ANOVA analysis, with maximum fold change based on spot normalized volume. P values of < 0.05 were considered statistically significant and reflect the probability that the observed change did not occur from random noise.
H. capsulatum viability test
To verify that the proteins found altered in our analysis did not result from the death of H. capsulatum, the organism was plated on solid medium following 24 and 48 hr growth with apo-transferrin. One hundred yeast were plated from cultures growing with or without apo-transferrin for 48 h. Eighty four colonies were counted on plates from H. capsulatum cultures containing apo-transferrin while 82 were counted from cultures free of apo-transferrin. Thus any changes we might find would be a direct response from the stress we were applying, and not from H. capsulatum death.
In-gel digestion
Protein bands were excised and diced into small pieces (~1 mm) and placed into 0.65 mL siliconized tubes (PGC Scientific). Each gel piece was briefly incubated and washed with 100 μl of water. Gel pieces were resuspended in 100 μl of acetonitrile and placed in a speed vacuum until dry. Next, 30 μL of 10 mM DTT in 25 mM ammonium bicarbonate was added to the dried gel pieces. Samples were vortexed and centrifuged briefly. The reaction was allowed to proceed at 56°C for 1 hr. The supernatant was removed and 30 μl of 55 mM iodoacetamide was added to the gel pieces. Samples were vortexed and centrifuged briefly. The reaction was allowed to proceed in the dark for 45 min at room temperature. Supernatant was removed and the gels were washed with 100 μl of ammonium bicarbonate by vortexing for 10 min. Supernatant was removed and gel pieces were dehydrated in 100 μL of 25 mM ammonium bicarbonate/50% ACN, vortexed for 5 min, and centrifuged. This step was repeated. Gel pieces were placed in a vacuum centrifuge until dry. Thirty microliters of a 12.5 ng/ml trypsin solution was added to the gel pieces and rehydrated on ice at 4°C for 10 min. Supernatant was removed, samples were centrifuged, and 25 μL of 25 mM of ammonium bicarbonate was added. Samples were centrifuged briefly and incubated at 37°C overnight. Following overnight digestion the solution was placed into a clean 0.65 mL siliconized tube. To the gel pieces, 50 μL of 50% acetonitrile/5% trifluoroacetic acid acid (TFA) was added. Samples were vortexed for 30 min, sonicated for 5 min, and the solution was combined with the first aqueous extraction. This was repeated and samples were then dried in a vacuum centrifuge.
MALDI-TOF mass spectrometric analysis
Dried peptide extracts were dissolved in 5 μL of water. Next, 2.5 μL of sample was mixed with 2.5 μL of alpha-cyano-4-hydroxycinammic acid in 50% acetonitrile/5% TFA, 10 mg/mL, and 1 μL of sample was delivered to a gold plated matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (MS) sample plate. Mass spectra were collected on a Voyager-DE PRO (Applied Biosystems, Foster City, CA) MALDI-TOF MS. Each spectrum was an average of 200 laser shots. Trypsin autodigestion peaks (2211.11 and 842.51) were used to calibrate each spectrum.
Raw MALDI-TOF data were processed using manufacturer-supplied Data Explorer software, with the following settings: Baseline correction parameters; peak width 32, flexibility .5, and degree .1 Noise filter/smooth was selected with a correlation factor of .7. Each MALDI spectrum was manually inspected to create a peak list. To control for peaks generated from keratin and trypsin, a spot was cut from an area of the gel containing no protein, and prepared with each sample. Peaks which were in both keratin/trypsin control and sample spectra were excluded from peak lists.
LC-MS/MS analysis
For nanoflow liquid chromatography (LC)-MS/MS analysis, dried peptide extracts were dissolved in 5 μL of 2% acetonitrile, 0.1% formic acid in water. Resuspended samples were loaded onto a Symmetry 5 μm particle, 180 μm × 20 mm C18 precolumn (Waters, Milford, MA), then washed 5 min with 1% acetonitrile in 0.1% formic acid at a flow rate of 20 μL/min. After washing, peptides were eluted and passed through an Atlantis 3 μm particle, 75 μm × 100 mm C18 analytical column (Waters, Milford, MA) with a gradient of 1–80% Acetonitrile in 0.1% formic acid. The gradient was delivered over 120 min by a nanoACQUITY UPLC (Waters, Milford, MA) at a flow rate of 250 nl/min, to a fused silica distal end-coated tip nano-electrospray needle (New Objective, Woburn, MA). Data were collected on a Q-TOF Premiere mass spectrometer (Waters, Milford, MA) set for MS survey scans and automatic data-dependent MS/MS acquisitions, which were invoked after selected ions met preset parameters of minimum signal intensity of 10 counts per second, ion charge state 2+, 3+, or 4+, and appropriate retention time. Survey scans of 1 s were followed by collision induced dissociation (CID) of the three most intense ions for up to 6 s each, or until 10,000 total MS/MS ion counts per precursor peptide were attained.
Raw LC-MS/MS data were processed using manufacturer-supplied ProteinLynxGlobalServer 2.2 software, with the following settings: Adaptive background subtraction of polynomial order 5 below a 35% curve, two smooths with a window of three channels in Savitzky Golay mode, followed by fast deisotoping and centroid calculation of the top 80% of peaks based on a minimum peak width of 4 channels at half-height. On the basis of these parameters, pkl files incorporating parent ion mass and peak lists for each corresponding MS/MS spectrum were generated.
Database searching
Protein/genomic databases 1, 2 and 3 were constructed beginning with the H. capsulatum G217B strain genome obtained from Washington University's Genome Sequencing Center website http://www.genome.wustl.edu/home.cgi. Database 1 was constructed using only Fgenesh and parameters setup by Softberry http://www.softberry.com/berry.phtml. Database 2 was assembled using publicly available EST libraries, also retrieved from Washington University's Genome Sequencing Center website http://www.genome.wustl.edu/home.cgi. EST's were generated from H. capsulatum cDNA under both mycelial and yeast phases, and assembled into full gene sequences with the aid of the contig assembly program (CAP)3 genome assembly program [29]. Database 3 was assembled using a combination of the gene finding programs Fgenesh, EAnnot, GeneWise, and SNAP [28, 30–32].
MALDI-TOF MS and LC-MS/MS data were used to search in-house databases 1–3 and publicly available fungal databases using MASCOT (version 2.1, Matrix Science, London, United Kingdom). A protein match from either of the 3 databases would count as a hit. Only protein matches deemed statistically significant (p < .05) by the MASCOT algorithm were accepted as protein sequence matches. For MALDI-TOF data, a 50 ppm mass accuracy threshold was set while a minimum precursor and fragment-ion mass accuracy of 1.2 and 0.8 Daltons was required for LC-MS/MS data. Up to 1 missed trypsin cleavage was allowed while fixed and variable carbamidomethyl were selected for separate searches. A decoy database was constructed by scrambling our in-house database sequences, and used to estimate false positive protein ID rates. Searching MS data against the decoy database did not result in any significant MASCOT scores. In order to annotate the protein sequence matches obtained from searching databases 1–3, each sequence was BLAST searched against the NCBInr sequence database.
Western blot analysis
H. capsulatum proteins prepared for 2D gel analysis were also probed using different antibodies specific for either cyclophilin or HSP 70. Ten micrograms of protein was mixed with 1× sample buffer (5×; 200 mM Tris-HCl, pH 6.8, 50% glycerol, 5% SDS, .5% bromphenol blue, and 5% beta-mercaptoethanol) heated at 95°C, and loaded onto a 12% SDS PAGE gel. Gels were run at 150 V for ~2 hrs. Following electrophoresis, gels were incubated in transfer buffer (25 mM Tris, 192 nM glycine, 15% v/v methanol) for 15 min. Proteins were transferred from 1D gels to nitrocellulose membrane overnight at 30V and 60V for 1 hr. Following transfer, each membrane was placed in 20 ml of blocking buffer for 1–2 hr. Blocking buffer consisted of 1× Tris-buffered saline (TBS) 5 g of non-fat dried milk/2.5 ml of 10% Tween. 10× TBS consisted of 24.2 g Tris base, 80 g of NaCl, pH 7.6. Primary antibody was added in a 1/5000 (v/v) ratio of antibody to blocking buffer and the membranes were incubated overnight at 4°C. This was followed by five 5 min washes in blocking buffer. Secondary horseradish peroxidase (HRP) conjugated antibody was added in a 1/1000 ratio of antibody to blocking buffer and the membranes were incubated for 1 hr. Membranes were then washed 5 times in 1× TBS for 5 min each. Immunoreactive bands were visualized using BioRad HRP color development reagent 4-chloro-1-naphthol and the AlphaImager 950 high-performance CCD camera. Spot densities were quantified using AlphaImager software.
2D gel analysis of H. capsulatum isolated from IFN-γactivated macrophages
Bone marrow derived macrophages were harvested 5 days following incubation with 10 ng/ml granulocyte macrophage-colony stimulating factor (GM-CSF). Macrophages were incubated for 24 hr with 5 ng/ml of IFN-γ. Leucine uptakes assays, as previously described7, showed that macrophages were activated to inhibit H. capsulatum growth by 80% (data not shown). Macrophages were infected with H. capsulatum at a multiplicity infection of 5 for 24 hrs. Following 24 hr incubation, macrophages were washed to remove any H. capsulatum that had not been ingested. Macrophages were then lysed in a buffer containing 19%, ethanol, .1% SDS, and 1% phenol. An H. capsulatum control was also prepared under the exact conditions except for the addition of macrophages. Cells were centrifuged at 2000 G's and the supernatant containing macrophage debris was removed. H. capsulatum cells were washed, lysed, and 2D gel analysis was performed as previously described. In order to retrieve an adequate amount of H. capsulatum protein approximately 3.0 × 107 macrophages needed to be infected for one biological replicate.
Using a proteomic approach we have begun to address the knowledge gap which exists regarding the identities and number of proteins regulated by H. capsulatum under host conditions. Among the H. capsulatum proteins whose regulation was influenced by lowering iron availability were metabolic enzymes involved in glycolysis, lysine biosynthesis, and the TCA cycle. Like S. cerevisiae and C. ablicans [37, 40], H. capsulatum may adjust its metabolism to more efficiently use the iron that is available. This modulation might be advantageous for survival in a nutritionally restricted environment. Recent studies have shown ferrous, but not ferric, iron maintains homoeostasis in H. capsulatum triacylglycerides indicating a role for lipid metabolism in relation to iron [27].
A shift in metabolism and a 2.0% reduction in protein abundance suggest that the organism is reducing the activity of expendable cellular processes to cope with environmental changes similar to those encountered in the host. Three of the H. capsulatum enzymes altered by iron levels; asparaginyl-tRNA synthetase, saccharopine dehydrogenase, and a protein with an amino acid sequence unique to H. capsulatum have yet to be connected to pathogen iron availability thus warranting further investigation.
We would like to thank Michael Wagner (Cincinnati Children's Hospital Medical Center) for mass spectrometric search engine setup and helpful discussions regarding statistical analysis. Thank you to Simon L. Newman for discussions pertaining to H. capsulatum growth assays. H. capsulatum sequences for database 1 were generated by the group of Elaine Mardis at the Washington University St. Louis Genome Sequencing Center, in collaboration with William Goldman (Washington University St. Louis) and Anita Sil (UCSF). This work was supported by NIH grants AI-42747 and AI-34361.
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Home » About University » Word by Mr. President of the University
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Word by Mr. President of the University
Dear sons/ students of Benha University
Dear brothers and colleagues of Faculties Members and Teaching Assistants
Dear employees in Benha University
Dear visitors of Benha university portal
In Benha University we are all working to enhance and develop the Egyptian human quality and skills through a scientific, research, academic, and social system. We have built a certain plan to achieve that aim. All the staff members, employees, workers, and under-post graduates cooperate to reach our goal of enhancing the Egyptian human qualities.
In Benha university we seek to spread and enroot the values of, tolerance, hardworking, morals, and Egypt belonging. In Benha University we aim to find and establish the value of home belonging that everyone should be proud of it (a home of a great and ancient civilization).
In Benha university we have a role in developing the community by providing it with appropriate hospitals- we always develop them regularly- to serve our local community and we try hard to equip them to be one of the most great and efficient hospitals in Egypt and the region.
In Benha University we are working to fulfill our community needs in several fields, so we are working to construct new faculties that will soon be real - in a few coming years.
In Benha university we try hard to provide the suitable educational, scientific, and research atmosphere and environment to our students to help them in their studying and creativity, so we always give a great interest to university housing campus and students activities.
In Benha University we in a race with time to achieve what we had promised Allah and Egypt to do.
President of Benha University


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In the Oregon Senate, most of the attention has been on the races for the Gresham seat held by Democrat Laurie Monnes Anderson and the open seat on the southern Oregon coast, but the money trail tells you the Senate Republicans are also making a big push to oust Sen. Mark Hass, D-Beaverton.
Although Hass's district has a 14-percentage-point Democratic registration edge and he has seemed well-entrenched, Republican challenger Gary Coe has now topped the $300,000 mark in fundraising -- compared to $137,000 for Hass (these numbers may not reflect current reality, since campaigns have seven days to report their receipts and expenditures).
As it happens, Coe, owner of a towing company, has topped that fundraising mark in an indirect way. Virtually all of his support -- $260,000 -- has been in in-kind contributions, mostly from the Senate Republican caucus fund. Essentially, the caucus political apparatus has been pouring big money into mail pieces and advertising to help Coe. Since Oct. 1, the Senate Leadership Fund has reported putting about $74,000 in the race.
Coe also reported receiving an in-kind contribution of $25,600 from the Oregon Family Farm Association for postage. So clearly, voters in Senate District 14 are getting plenty in their mailbox these days.


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- Calories 237
- Caloriesfromfat 18%
- Fat 4.7g
- Satfat 0.7g
- Monofat 3.3g
- Polyfat 0.5g
- Protein 4.2g
- Carbohydrate 45.6g
- Fiber 3.6g
- Cholesterol 0.0mg
- Iron 2.6mg
- Sodium 600mg
- Calcium 34mg
No Boo-Hoo Baked Potatoes
Photo: Randy Mayor
How to Make It
Step 1
Prepare grill.
Step 2
Cut 1 potato lengthwise into quarters; place on a 12-inch square piece of foil. Brush cut sides of potato with 1 teaspoon oil. Sprinkle with 1/4 teaspoon basil, 1/4 teaspoon salt, 1/8 teaspoon oregano, and a dash of pepper. Top each with 2 tablespoons onion and 3/4 teaspoon garlic. Reassemble potato; tightly wrap potato in foil. Repeat procedure with remaining potatoes and ingredients. Nestle potatoes in coals or on briquettes; grill for 1 hour or until tender, turning every 15 minutes.
Step 3
Note: To prepare potatoes in a conventional oven, preheat oven to 475º. Place foil-wrapped potatoes on bottom rack in oven. Bake at 475º for 1 hour or until tender.


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The Marketing area of interest develops the understanding and skills necessary to manage the process by which individuals and organizations obtain what they need and want through the exchange of ideas, products or services.
Students interested in marketing benefit from outstanding teaching and courses in marketing communications, research, strategy and product design (including the rapid prototype lab) that earned Willamette recognition from Princeton Review as "one of the 15 best programs" in the U.S. for preparing students for careers in marketing. Students also benefit from the professional, leadership and networking opportunities available through our student chapter of the American Marketing Association.
Job titles of alumni in marketing careers include director of marketing, public relations manager, account executive, marketing analyst, marketing research manager, e-business marketing manager, consultant, sales and marketing manager, product manager, and vice president.
The elective courses listed below are recommended for students interested in Marketing. Students may select courses from the list and from other areas of interest to build a customized individual program that meets their specific goals.
Elective courses in each area of interest are offered every year. It is possible that a specific course may not be offered in a given year and that previous courses will be replaced by new courses.
Recommended Elective Courses
Students may select courses from the list and from other areas of interest to build a customized individual program that meets their specific goals.
- GSM 6216 - Business and Economic Forecasting
- GSM 6231 - Strategic Marketing for Public and Private Not-for-Profit Organizations
- GSM 6241 - Industry Analysis
- GSM 6243 - Sales Force Management
- GSM 6249 - Project Management
- GSM 6260 - Research for Marketing Decisions (Highly Recommended)
- GSM 6261 - Marketing Strategy (Highly Recommended)
- GSM 6262 - Integrated Marketing Communication (Highly Recommended)
- GSM 6269 - Value Chain Management
- GSM 6272 - Data Sciences for Strategic Applications
- GSM 7251 - Internship


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Plan of Salvation
Simply put, Mike Washer loves God and teens. He is a gifted preacher and a careful expositor. His faithful ministry to the young people at Baptist Church has been Holy Spirit blessed and there is much fruit that remains. As his home church, we share his burden for evangelism beyond the boundaries of Spartanburg, SC. Westgate Baptist Church has pledged her support and is privileged to be an ongoing part of his growing ministry. I recommend him to you without reservation and I am confident that he will help you reach your community for Christ.
Youth evangelism is a needy field that is white unto harvest. The Lord has burdened Evangelist Mike Washer to help meet that need. As a youth pastor, the Lord has used Mike to nurture a youth group that is growing spiritually, and burdened him to reach the lost. One of the effective outreaches of their calendar has been their basketball outreach. The Lord has used this outreach to reach scores of young people with the Gospel. Now God has laid upon his heart to take this evangelistic tool on the road. Mike has a heart for souls and for youth. I recommend him to your consideration.
Mike Washer has preached summer teen camps and participated in various programs here at The Anchorage. His preaching is Biblical, passionate, powerful, and practical. His conviction on the power of the Gospel and the authority of Scripture has been used by the Holy Spirit to produce noticeable conviction in many, resulting in spiritually changed lives. I have found him to live as he preaches, showing genuine care and compassion to those around. His wife is nothing but a credit to him and his ministry. I heartily recommend his ministry to you.
"My observation and participation in the ministry of National Hoops has been really encouraging. It is well-organized, ethical, and God-dependent. The Lord has already used it to bring many young souls to Himself. I especially appreciate its separated stand, biblically solid follow-up materials, and carefulness to be a servant to local churches. The Philippines needs ministries with ingredients like National Hoops."
Dr. Phil Kamibayashiyama, BJMBC Director, Metro Manila, Philippines
Copyright 2009 | NationalHoops


text:
For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
Full length native protein (purified) (Pig).
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The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Use at an assay dependent concentration.
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|Use at an assay dependent concentration.
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ab6322 at 1/100 staining rabbit oesophagus tissue sections by IHC-Fr. The tissue was paraformaldehyde fixed and blocked with serum before incuabion with the antibody for 1 hour. An Alexa-Fluor ® 594 conjugated goat anti-mouse IgG1 was used as the secondary.
IHC image of Desmin staining in human skeletal muscle formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab6322, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"


text:
Blackguards 2 Discussion
Trainer Tools and Resources
With the game's full release on Steam now, it seems the trainer does not work with the Steam version.
Can anyone confirm if they managed to get it to work with the legit Steam version?
You can use the game built in console.
Just add -devcon to your launch options, or shortcut.
You got cheats like "godmode".
"help" should show you all commands.
Though I already found it by searching around.
And cheated the AP with CheatEngine as well.
gettin real depressed here ...can somebody help?
[Edited by Madmenace, 1/22/2015 8:11:42 AM]
edit: the dev. console helped
[Edited by Madmenace, 1/22/2015 8:14:08 AM]
[Edited by tbenzin, 5/31/2015 8:05:04 PM]
I have a lil prob with the trainer ! Everytime i try to activate it, i got an error " A pipe with the specified name is not open on the target machine". I've opened the trainer in admin mode, and my anti-virus is disabled, so why does it not work , Any help is welcomed ! :c
* Updated game trainers and cheats daily
* Get notified when new cheats are added
* Request which games get new trainers
* Priority support with any problem
firewall and other security software.
on trainer and game.
version and distribution.


text:
Monday, April 2, 2012
Cherry Blossom 10 Miler: race report
I tapered for the race by doing my longest and hilliest bike ride of the year to date and then trashing myself on a big pile of 100s in the pool the day before. Instead of going to bed early, I had a complete meltdown on the living room floor in my underwear, trying to get the pedals off my bike. I didn't even lay out my clothes in little piles and take a picture of my bib number the night before. Somehow I woke up early enough to walk out the door with everything I needed (except a thin and even coat of body glide). Despite the hot mess of my race prep, I was actually looking forward to the race. A MAF test does not (should not?) hurt, and I was anticipating running ten relaxed and perfectly precise miles.
We got down to the mall early enough for me to do my warm-up and get settled. I actually really like the pre-MAF test warm-up. I start out walking and let my heart rate climb up only a few beats per minute for two miles, hoping to end somewhere within five beats or so of MAF. The other end of the mall was quiet and it was nice to just relax into the sound of my feet scrunching on the gravel and breathe. However, once my HR got to the low 140s, it didn't want to budge. I came flying back down towards the bench where the poet was parked, and I think I managed to get it over 145 for just a few seconds. There's a fatigue/fitness correlation that I still don't quite understand, but what it generally means is that when I'm fatigued, I have to run my sweet sweet ass off to get up to MAF. Crap.
I climbed up the side of a light pole to look for my friends, and finally found Liz and her sister. Side note: taking perfect race pictures apparently mutated genetic abnormality.
We had talked earlier in the week about starting together, but I warned Liz that I wouldn't be able to snort or giggle or chat because all of those things eat heart rate. While we were lined up, I warned them that the opposite might be true. Our corral was extremely crowded, and we shuffled forwards and were suddenly across the line. Every race I've ever run with crowded corrals usually thins out by the end of the first mile, and like I said, I've never run this race so I had no idea what to expect. We started running, and I tried to haul up my heart rate, and when it wouldn't move, I started chattering non-stop about fitness and science and fatigue and bicycles in hopes that I could cheat out some extra beats. Here we are about 1/2 mile in. Holy ridiculously crowded race.
When I lapped my watch at mile one and saw that my heart was still in the 130s and how slow we were running, I decided to try and pass a bunch of people in hopes of getting to a clear spot where I could run hard enough to get my heart rate up. I spent the next nine miles passing people, looking for this spot, and never found it. The race was just as crowded at the finish as it was in the starting corral. This is roughly mile five.
Around mile three I realized that this might be the case and just decided that I wouldn't get upset about it, I'd just do my best to keep my heart up and enjoy the morning. My hamstring that's part of my pissed-off-posterior-chain started grumbling around mile four, so I started cycling the loop of form questions in my head over and over. The only places where the crowds were really a problem was in the water stops around mile six and eight, where I was actually forced to walk for 30-40 feet because there was simply no way to get through. Around mile seven I started to feel a bit tired, and annoyance at my uneven splits and the crowds really crept in. We were running around Hains Point at that point, and people were running across the grass and down onto the sidewalk by the river, just to have a clear space to run.
Usually what happens at the beginning of the last mile in a MAF test is that I flip my watch from the HR-only screen over to the screen that has my average for the run. I do this because sometimes I need to run a tiny bit harder to bring up my average. When I flipped over my screen at mile nine, my average HR for the run was 145. I don't have enough HR in my body to bring that up to my MAF average in one mile, but I did make sure that last mile was the fastest of the day.
And then I was done. The post-race corrals forced you to walk about two blocks until you could get out, and then I jogged very slowly back to where I was meeting the poet to cool down. My legs felt a little more tired than a long run but not nearly as tired as a hard race effort. And I got several shots during the race of some pretty decent form, including this prizewinner from Cheryl. However, this picture plus the fact that I have a 6-inch chafe scab on my left thigh means that I am throwing these CW-X shorts in the garbage.
I'm glad I ran this. I'm glad that I can now lift the voodoo off of yet another race and file it away, but until I'm fast enough to get a seeded spot in this race (aged 60?), I don't think I would run it again. Trying to PR in these conditions would be rough. Some of my friends who started in faster corrals managed to PR and have great days, so maybe the trick is to be extremely ambitious about my "estimated" finishing time, but regardless, I don't think this is for me. The last three half marathons I've run have been enormous races - certainly larger than this one - but they've been run on streets large enough to not throttle the traffic down, and that is what this part of DC lacks. I'm not sure if I'll be running the GW Parkway ten miler in a few weeks or not, but I know that it doesn't have this problem.
And now that I've run two mid-distance races extremely fatigued on no taper, I'm starting to get very curious about what my rested fitness looks like. I have no problem with the way I've executed either of these races, but all the aerobic training I've been doing is starting to poke out of the mush at me. Despite the calf setback and the fact that I basically feel like I'm the disastrous red-headed step-child of my coach most of the time, progress is being made. I might not get a chance to see what this rested fitness looks like until my 70.3 next month, and a 70.3 is certainly a very different animal than a stand-alone running race so who knows what will happen, but indeed. Curious.
25 comments:
I also ran this race yesterday and wrote a blog post about it, basically saying the same thing you said. It was a good experience for the most part but I don't think I would do it again. I just felt too frustrated and stressed out by the crowds that never thinned out. It made it difficult for me to enjoy myself and try to run fast. But congrats on a well executed race and for lifting the curse! :)ReplyDelete
I've run a couple of races like that where the masses just never seem to thin out. I have no patience for it if I'm trying to 'race' by any means. Glad you managed to have some fun out there and put in some good effort. Congrats!!ReplyDelete
OMG the people!!!!!!! I would have stabbed the hell outta some people. Good lord.ReplyDelete
Congrats on surviving that shit!
I read this whole report not knowing what MAF is. I still don't. But it sounds like you gave it a great effort & did a wonderful job. That much I do know.ReplyDelete
What I notice when I look at these photos is how entirely happy you look!!! And I love that.ReplyDelete
Holy crowded!ReplyDelete
Lovely photos of you & that a$$! ;) Seriously, looking strong, my friend!ReplyDelete
How did I miss that you were doing this?! Dangit. I was in a higher "seed" and still found it nearly impossible to go fast. So, that's just this race's MO. Crowds, usually no more blossoms, but a course we keep coming back to nonetheless.
Love all the smiling in the photos -- it looks like you are having a great run and not actually wanting to stab the 15,000 people in a 10-inch radius around you, which is what I most certainly would have been doing.ReplyDelete
I'm glad you were able to remove another race from the jinxed pile!ReplyDelete
So uplifting to see your big smile and have you running happy!ReplyDelete
Road races have gotten insanely crowded. Dodging people takes an amazing amount of energy and makes racing (full out) impossible. I enjoyed the Army 10 Miler a couple of years ago because I'd been injured, and unable to run, and had no goals or intentions. But I don't think I'd ever sign up to race it.
Great job with the Where's Waldo in that first photo! :-) So happy to hear that you had a good race and you finally got to experience the Cherry Blossom awesomeness!ReplyDelete
You look like you were having a GREAT time during this race! The pictures are awesome!ReplyDelete
Your photos from the race are great, even if the crowd wasn't. At least you got a chance to experience the race. Even if you don't do it again, you've lifted the curse.ReplyDelete
It was ridiculously crowded. But I had fun and it was fun to see you again however briefly! I didn't realize that was Liz who I did meet last year.ReplyDelete
Sorry that I wasn't able to find you guys! Based on my experience, I wouldn't have been able to stay with you for long. Congrats on having a good race!!!ReplyDelete
Monkey - back - GONE.ReplyDelete
It was a pretty well-organized event, all things considered, but you're right that it's run on some of the tightest streets you can find downtown, not like PA Ave or Independence that have up to six full lanes when closed to traffic. I'd do it again but maybe as a warmup for the Charlottesville half which is next Saturday.
Congrats on finally being able to do this race. I've heard a variety of things about this race, but those pictures of the crowds are nuts!ReplyDelete
Nice! I'm glad you had a good time and I would much prefer to run a MAF test like this than around and around in circles. Also, I thought it was very interesting to see your "MAF method." I've never thought of watching the HR average for the entire time (duh). I usually watch actual and lap average. Storing that away for next time.ReplyDelete
Holy schmee, croweded race! I'm glad it was at least a decent race. I've had a few races as such and it's nice to see someone else conquering them...now maybe I will. :)ReplyDelete
So glad things are finally coming together and making sense. It's a good feeling :)ReplyDelete
And one Cherry Blossom was more than enough for me, what a hot mess.
You looked so happy every time we saw you! I'm also glad that Thom captured my back in the starting picture.ReplyDelete
Do you run all your races with that smile plastered across your face? Or do you just smile for cameras?ReplyDelete
I've heard that Cherry Blossom is a super crowded race and tough to PR in. I would have PRed though because I've never raced a 10 miler before.
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In either case, the bank will desire to know how you prefer to execute your plan of action nie.im most lenders will likely be happy to assistReplyDelete
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I have to say...I love this race and the crowds don't bother me. For some reason they pump me up. Perhaps I sound too Pollyanna, but I spent the first 5 miles passing people...and then the next five miles passing more people -haha I love it!!!ReplyDelete


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MisraBani TantriCharya–Varanasi
The first MisraBani TantriCharya is proposed to be organized at Varanasi in October 2013 with collaboration of cultural organization(s) at local/ national level. Benares Hindu University has kindly offered its facilities for organizing Tantricharya. The event shall be held at Academic Staff College from 12 to 18 and at Faculty of Music and Performing Arts no 19th and 20th October 2013.
Dr. Lalmani Misra
Dr. Lalmani Misra, well known as a Vichitra Veena player, had an extraordinary ability for creating music. Spontaneity and perfection defined his music. The following remarkable quotes by prominent artists and members of the musico-academic community would give a brief notion of Dr. Misra's exemplary creative strength before we proceed to analyze his genius:
"Prof. Misra was the Music Director of Udaya Shankar's troupe and job amply speaks of his eminence as musician. He contributed some of lively tunes and melodious notes to music performances given in foreign countries which kept the audience spell bound. He gave his performance in almost 150 big towns of America and Europe and was hailed by one and all as a talented artist of the age. Prof. Misra can play on eight instruments with a master's hand." [News Paper item (Telegraph May 6, 1952) excerpt]
Dr. Misra's contribution to the music world is that he has written hundreds of compositions for sitar in more than 150 common and uncommon Ragas. Moved by a desire to perfect a system in instrumental music that would be as complete as the vocal tradition, he created a new Gat style, which gradually has come to be known as 'Misrabani'. It was also referred to as 'Koot Ki Gat' owing to the complexity of the Gat. They were so labeled, recalling instances of using such complex rhythmic patterns by earlier musicians in flourishes hailed by the aficionados as Koot ki Taan.
The Misrabani compositions he created are serious compositions in stylistic sense. They clarify that selection of Raga-s influences the minds of musicians.
Under this style he created a new Gatkari in Vilambit Jhoomara Tal, Vilambit Jhap Tal and Madhya-laya Ada Char Tal. In this new style Dr. Misra introduced Mizrab Bol DA RDA -R DA. Especially, in vilambit pace Da Rda -R Da shows a very different dimension of manifestation.
The oblique movement and complexity of a Raga is considered one of the main reasons for its unpopularity. There are many Raga-s that are extremely melodious and fulfill all the desired norms of Indian classical music (proper consonances with lower and upper tetra-chords) but owing to their complex nature they are rarely played by the instrumentalists. Most of such demanding Raga-s like Basant Bahar, Gandhari, Malhua Kedar, Anand Bhairav, Multani, Kaushik Kanhada, Malgunji, Bageshri Kanhada, Sindoora, Neelambari etc. were played by Dr. Misra.
Workshops have been engaged by Dr. Ragini Trivedi around the country to train Sitarists in this advanced and cerebral style of playing. One may view few excerpts on Youtube channel of Omenadnet.
MisraBani TantriCharya is aimed at young instrumentalists who have received professional training for 5 years or more.
Learners in the group (10 to 15) would be trained by two Misrabani Sitarists under supervision of Dr. Ragini Trivedi, daughter of Sangeetendu Pt. Lalmani Misra, creator of Misrabani string style. Senior-most disciple of Dr. Misra, Dr. Pushpa Basu would give expert guidance.
MisraBani TantriCharya would be hands-on intensive workshop of two four- hour sessions each day for 7 days.
- Learners would be provided detailed syllabus with Mizrab Bol-s, Alankar-s and Compositions in play-as-you-read digital notation system, Ome Swarlipi.
- Learners would make their own arrangements for lodging & boarding.
- Learners would bring their own instruments: Sitar/ Sarod/ Veena/ Santoor.
Interested learner may send in their application forms through email and hard copy.
Email should be sent to firstname.lastname@example.com with subject; MisraBani TantriCharya attaching filled in application form by 15th August 2013 and scanned image of Demand Draft. The hard copy should be sent to Astha, B-31, Aakriti Garden, Nehru Nagar, Bhopal – 462003 (M.P.) India so as to reach before 31st August 2013.
Registration Fee:
Till 30th June 2013 Rs. 3,000.
Till 31st July 2013 Rs. 4,000.
Till 31st August 2013 Rs. 5,000.
Till 30th September 2013 Rs. 9,000 provided seats are available.
Discount for Students* Rs. 1,000.
A limited number of Sponsorships by disciples of Sangeetendu Pt. Lalmani Misra have been reserved for exceptional learners between 21 to 28 years pursuing regular course in music in college/ UTD, subject to their selection by Madhukali.
Those desirous of Sponsorship should submit a detailed note of recommendation from a teacher of repute. The note should detail particulars of Raga, Tal the candidate can perform flawlessly. Application form & detailed note with Demand Draft of Rupees 1000 only payable to Madhukali at Bhopal should reach Astha, B-31, Aakriti Garden, Nehru Nagar, Bhopal – 462003 (M.P.) India before 15th June 2013. Scanned image of Demand Draft and duly signed note of recommendation, along with application in pdf (Portable Document Format), should be emailed to firstname.lastname@example.com before 10th June 2013.
*Students of UGC recognized College and University Teaching Departments would have to present current Scholar Identity Card along with recommendation of Head of Department.
Click here to download form.
Articles on Omenad
Video on Omenadnet
Tweets #Tantricharya Follow @madhukali and @omenadnet
Misrabani Vichitra Veena: heritage aLive Vol. 1 {Published}
Format: Sound Recording
Year: 2013
Publisher: Misrabani, Philadelphia, 2013
Description 1 audio disc (52 min.) : digital ; 4 3/4 in.
Raga Malgunji 38:50
Alap, Jod
Vilambit Jhumra Tal (slow 14 beats)
Drut Ada CharTal (fast 14 beats)
Raga Bhupali 12:50
Kaharwa Tal (8 beats)
A Dawn of Veena: Bairagi on Vichitra Veena by Dr. Lalmani Misra {more}
Format: Sound Recording
Year: 2013
Publisher: Madhukali, Bhopal, 2013
Description 1 audio disc (51 min.) : digital ; 4 3/4 in.
Raga Bairagi
Alap: 19:33
Jod: 10:24
Gat: 11:20
Raga Bhairavi
Dhun: 05:22
Celestial Music of Pandit Lalmani Misra: Vichitra Vina {more at omenad}
Format: Video
Year: 2006
Publisher: Rohnert Park, CA: Music Dept., Sonoma State University, 2006
Description: 1 video disc (45 min.) : sd., col. ; 4 3/4 in.
Raga Sindura
Alap, Vilambit & Drut teental: 31:28
Raga Tilang 10:23
Raga Bhupali 15:31
India: Vicitra Vina : The Music of Pandit Lalmani Misra = Inde : Vicitra Vina : La Musique De Pandit Lalmani Misra {buy at amazon?}
- Format: Sound Recording
- Year: 1996
- Publisher: France: Auvidis Unesco, 1996
- Description: 1 sound disc (62 min.) : digital ; 4 3/4 in.
- Tracklist
Raga Kausi Kanhada (Malkauns style)
Gat in slow tintal
Gat in fast tintal
Nectar of the Moon: Vichitra Vina Music of Northern India {buy at amazon?}
- Format: Sound Recording
- Year: 1981
- Publisher: Los Angeles: Nonesuch, 1981
- Description: 1 sound disc : 33 1/3 rpm, stereo. ; 12 in.
A Raga Ananda Bhairava 20:04
B1 Raga Multani 12:16
B2 Dhun In Raga Ananda Bhairavi 7:54
Material in possession of Libraries (Worldcat)
Publisher: Los Angeles, Ca. : Ravi Shankar Music Circle, ©1979
Document Type: Sound Recording
All Authors / Contributors: Lalmani Mishra; Chotey Lal
OCLC Number: 11153496
Performer(s): Lal Mani Mishra, vichitra veena ; Chotey Lal, tabla.
Production notes:
"Recorded in concert, Herrick Chapel, Occidental College–3/18/78"– Container.
Description: 1 sound cassette (60:00) : stereo.
Basant bahar in Ektal, 12 beats and Teental, 16 beats —
Bhairavi in Dadra, 6 beats —
Tabla solo Delhi style in Teental, 16 beats.


text:
So because I sort of have a slight addiction to horrible TV, I decided to check out this new show called Catfish. Oh, it's terrible. Entertaining, but terrible. At the beginning of every episode I can't help thinking to myself, "This is going to end badly." Basically people who are in online relationships, but have not met and/or have suspicions about the person they are in a relationship with (and I use that term very loosely) contact this dude and he goes and investigates. Then they meet and it's either really awkward or really creepy. I'm watching my second episode and so far I've learned two things. One - there are a lot of lonely people in this world. And two - people are batshit crazy. I actually find myself making bets whether these people are going to end up being who they say they are. Its highly exploitative. I also find it very suspicious that these people take the deception, so well. The girl I've been talking to intimately is really a dude? Well gosh darn it. The guy I'm now engaged to is really a woman? Well I love her anyway. If this was the real world, these meetings would be much angrier and I suspect in many cases, violent. So thank you, MTV for once again proving there is no reality in reality tv. What the hell is wrong with these people?? And how is it there are enough of them out there to make a show?!
Fellow humans! Listen up! Stop what you are doing and go find your bullshit meters! Somewhere in the middle of tweeting and Facebooking, you lost them. Back in the days before social media, (you know when the majority of conversations occurred in person) most of us were able to tell if someone was selling us a load of shit. If what a person was saying didn't make sense, little red flags would go up. Now you think it's perfectly normal to share your deepest, most intimate thoughts with a person you have never met, who could turn out to be a complete sociopath. Have you never seen an episode of Criminal Minds? Law and Order? The Crying Game? If you are talking to someone online and they never want to talk to you on the phone, or never want to meet you face to face, run! If they are out of your league, be suspicious! Seriously, if you are average looking and "dating" someone who looks like a swedish bikini model, chances are extremely good that the bikini model is some guy who looks like Eric Cartman. For crying out loud, use some portion of your brain. Then we wouldn't have a show like this and we could go back to watching Teen Mom and Real Housewives.
Monday, January 28, 2013
"What's on my TiVo?" Catfish edition.
Posted by S at 1:47 AM
Labels: Catfish, reality tv
No comments:
Subscribe to: Post Comments (Atom)


text:
Carmen Erkelensfrom
Carmen Erkelens enrolled in a Essentials of an Online Course course in 2016
This online course has been an eye-opener for me.
How did you experience the course?
This online course has been an eye-opener for me. I've learned a lot about teaching online in general, all the different learning tools and the basics of creating a course design. The experience of being in an online course and being part of a learning community is also very useful. It showed me that it takes both time and effort from the participants and the e-moderator. I'm looking forward to further develop my course design and to try out the different learning tools.


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Don't miss stories. Follow Raw Story!
How can humanity reorganize society to harness technology, eliminate money, manage natural resources and still provide for all? That tough question has plagued social engineer Jaques Fresco since he was a boy. Today, he preaches "technical revolution," traveling the world promoting his "Venus Project" vision for a remade society. What is the "Venus Project"? Here, it's explained …
This video is from FutureByDesign, published Oct. 7, 2008.
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Man arrested over match-fixing claim in Bulgaria
A former player from Bulgaria's top flight has been arrested over allegations of match-fixing involving the national under-19 side.
The Bulgarian Football Union (BFU) released a statement on Friday, confirming the arrest of a 20-year-old man.
It read: "Joint operational work of the interior ministry and the Bulgarian Football Union has led to an arrest for attempted manipulation of the national under-19 team matches.
"As a result of operational investigative actions, numerous individuals were questioned, including current under-19 internationals, who participated in the finals of the European Junior Championships in Hungary.
"These players are players of elite clubs from the top flight, which required information and assistance from their clubs.
"The Bulgarian Football Union would like to express its gratitude to those clubs to maintain the confidentiality and [to those who] cooperated fully in the investigation.
"We regret to note that the rapid actions had to be taken as a result, [which] probably influenced to some extent in purely psychological terms, the state of the national team during Euro 2014.
"The Bulgarian Football Union, however, is determined to do everything in their power to eradicate completely the manipulation of matches in the country."