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We present an optimized probe design for copy number variation (CNV) and SNP genotyping in the Plasmodium falciparum genome. We demonstrate that variable length and isothermal probes are superior to static length probes. We show that sample preparation and hybridization conditions mitigate the effects of host DNA contamination in field samples. The microarray and workflow presented can be used to identify CNVs and SNPs with 95% accuracy in a single hybridization, in field samples containing up to 92% human DNA contamination.
Plasmodium falciparum is the intracellular parasite responsible for the majority of the world's malaria morbidity and mortality burden in humans, causing an estimated 243 million episodes of malaria and 863,000 deaths each year [1]. Efforts to control and eradicate malaria are hampered by the accelerated evolution of drug resistance in the parasite. To date, the parasite has developed resistance to all major antimalarial drugs, raising concerns about the spread of drug-resistant parasites and the ability to effectively treat malaria [2]. The development of new technologies aimed at understanding parasite genome variability provides hope in identifying new drug targets, implementing smarter treatment plans, and ultimately reducing or eliminating the burden of malaria.
Genome variation such as SNPs and copy number variation (CNV) underpins P. falciparum drug resistance. The primary determinant of chloroquine resistance is a mutation in the P. falciparum chloroquine resistance transporter gene on chromosome (chr) 7 [3,4]. In vitro resistance to the antifolate drugs sulfadoxine and pyrimethamine increases in a step-wise manner as mutations accrue in dihydrofolate reductase and dihydropteroate synthase [5-8]. Varying copy number of the P. falciparum multidrug resistance 1 gene on chr 5 influences parasite susceptibility to a range of antimalarial drugs, including mefloquine, lumefantrine, quinine, and artemisinin [9-11]. Amplification on chr 12 of GTP cyclohydrolase 1 of the folate biosynthesis pathway is correlated with antifolate drug resistance [12,13]. These examples emphasize the importance of genomic variation in drug resistance and need to assay both SNPs and CNV genome-wide in the malaria parasite.
Microarrays provide a relatively fast and inexpensive way of examining genomic variation in P. falciparum [14]. Array comparative genomic hybridization (CGH) has been successfully used to look at structural variation and CNV in multiple P. falciparum strains [12,15-17], while large-scale sequencing efforts identifying SNPs [18-20] have spurred the development of SNP microarrays. Neafsey et al. [21] genotyped 1,638 out of 3,000 queried SNPs with 100% accuracy using an Affymextrix 3K SNP assay. Mu et al. [22] used Affymetrix molecular inversion probe technology to genotype 2,763 of 3,354 SNPs with >90% call rate. Multiple groups have successfully applied CGH for SNP detection with 80 to 90% sensitivity to approximately 3,000 SNPs [23,24] and identified parameters influencing SNP detection [24,25]. However, the reported detection rates are based on a core subset of SNPs (approximately 3,000) in a genome with more than 100,000 cataloged SNPs (PlasmoDB v5.5) [26].
Our central goal was to develop a single microarray platform that can assay CNV and genotype SNPs simultaneously and to optimize this platform for the challenges of monitoring monoclonal patient blood samples from field studies. We first empirically determined the optimum probe lengths and melting temperatures for SNP genotyping in the 81% AT P. falciparum genome. This was used to guide the design of a single high-resolution genotyping microarray with variable length probes optimized for high quality SNP genotyping and CNV detection (Figure 1). One half of the microarray interrogates 45,524 SNP loci using optimized resequencing probes 29 to 41 bp in length capable of making a base call at a precise nucleotide position [27]. The second half identifies CNV using tiled CGH probes 50 to 75 bp in length. We determine the reliability and accuracy of the CNV-SNP array using the laboratory lines 3D7, HB3, Dd2, SC05, and 7C126. We then validate the utility and robustness of the microarray using field samples with limited parasite DNA and high human DNA contamination present using blood collected from humans at the Thailand-Burma border.
Figure 1. Microarray layout and design. The microarray contains blocks of probesets for SNP genotyping and CGH. SNP genotyping probesets are composed of two probe quartets, one for each strand (red blowouts). Probes from one quartet typically have hybridization signals in a similar dynamic range. These signals are used to determine a base call and calculate base calling robustness, which is expressed as a Dscore where the two lesser hybridization signals are used to estimate background noise for each probe quartet (grey inset). The CGH probesets record data that are used to generate log2 ratios of a test and reference sample.
Effects of probe length and probe melting temperature on the robustness of base calls
Using a prototype 5K SNP chip, the performance of static probe lengths was compared to the performance of variable length, isothermal probes on base calling robustness. A base call is considered robust when a probe quartet has a single high nucleotide signal relative to the other three nucleotide signals, and the sense and antisense base calls are complementary. A Dscore calculating the background noise relative to the highest signal intensity in a probe quartet (Figure 1, grey inset) was used to compare the performance of static probes and isothermal probes. A Dscore close to 1 indicates high background noise and poor discrimination ability between probe signals, while a score close to 0 indicates low background noise and good discrimination ability. The mean Dscore of the 5K SNP chips was plotted for static probe lengths (Figure 2a) and for probe melting temperature (Figure 2b). Statistical analysis with a one-way ANOVA indicates significant differences between mean Dscore at various probe lengths (P < 0.0001), and a Tukey's multiple comparison test indicates that all probe lengths except 39- and 41-mers have a significantly different mean Dscore (P < 0.05). Out of the nine tested probe lengths, 39-mer probes generated the lowest mean Dscore with the best discrimination ability ( = 0.3575). A one-way ANOVA analysis indicates probe melting temperature Dscore are significantly different from one another (P < 0.0001), and a Tukey's multiple comparison test indicates melting temperature in the 66°C range was significantly different from other melting temperatures (P < 0.05). The lowest mean Dscore with the best discrimination ability for probe melting temperatures was generated at 66°C from the range of 42 to 82°C ( = 0.2647), and this outperformed any static probe lengths. Similar performance was seen when comparing exons, introns, and intergenic regions (Figure S1 in Additional file 1).
Figure 2. Base calling robustness is affected by probe length and melting temperature. Mean Dscore is plotted by (a) probe length and (b) probe melting temperature where vertical lines indicate 95% confidence intervals. Lower discrimination scores indicate greater base calling robustness. Fixed length 39-mers provided the best performance for any static probe length tested. Probes with a 66°C melting temperature provided the best performance for any melting temperature range and surpassed the performance of 39-mer probes.
Additional file 1. Supplementary Figures S1 to S3. Figure S1: pptimal probe melting temperature is consistent in exons, introns, and intergenic regions. Mean Dscore is plotted by probe melting temperature in exons, introns, and intergenic regions; vertical lines indicate 95% confidence intervals. Probes with approximately 66°C melting temperature consistently provided the best performance. Figure S2: CGH data reproducibility. CGH scatterplots for individual CNV events are displayed for replicate hybridizations from independent labeling reactions demonstrating data reproducibility. CNV events from three separate parasite clones are displayed: (a) Dd2; (b) HB3; (c) SC05. The CNV breakpoints are precisely identified between hybridizations. Figure S3: CNV detection in a WGA field sample. CGH scatterplot for a CNV event detected in a WGA field sample, M1064. Four genes (PFE1150w, PFE1155c, PFE1160w, and PFE1165c) are affected by this CNV, including the P. falciparum multidrug resistance gene, pfmdr1.
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Microarray base calling accuracy
Microarray data for 3D7, HB3, Dd2, SC05, and 7C126 (n = 15, 5, 3, 2, and 2, respectively) were compared to genome sequence data to ascertain base calling accuracy (Figure 3a). A useable base call was made at a SNP locus when the sense and antisense probesets indicated complementary bases; otherwise, there would be no base call for that SNP locus. Figure 3a depicts mean microarray accuracy plotted by Dscore; also depicted are CNV-SNP array data for the SNP subsets that are represented on SNP genotyping microarrays developed by the Broad Institute [21] and NIH [22]. The CNV-SNP array genotypes 1,507 of the 1,631 publicly available Broad Institute SNPs and 2,621 of the 2,743 publicly available NIH SNPs. For all SNP sets, a lower Dscore is associated with higher accuracy. SNPs from the Broad Institute and NIH maintain >95% accuracy at all Dscore cutoffs with 97.1% accuracy and 98.7% accuracy, respectively, at a Dscore cutoff of 1. The accuracy of all SNPs assayed on the CNV-SNP array maintain >95% accuracy for Dscore ≤0.9 but drops to an average accuracy of 94.6% at a Dscore of 1. Figure 3b depicts the mean number of base calls made by the microarray plotted by Dscore for all SNPs and for the SNP subsets from the Broad Institute and NIH microarray platforms (Figure 3b). On average, a microarray hybridization yielded 36,948 base calls from the 45,524 assayed SNP loci for a base call rate of 81.2%. The Broad Institute and NIH SNP subsets had an average call rate of 90.7% and 93.0%, respectively. Lower numbers of base calls are made at more stringent Dscore cutoffs for all data sets. When comparing accuracy for SNP subsets in exons, introns, and intergenic regions, introns and intergenic regions perform similarly, and exons exhibited the best performance (Table 1).
Figure 3. Microarray accuracy rate and base calling. (a) Microarray base calling accuracy rate was calculated by comparing microarray base calls with sequence data for five parasite genomes. At more stringent discrimination scores, accuracy rate increased. SNP subsets demonstrate greater performance than average if those SNPs are more amenable to hybridization-based interrogation (NIH and Broad subsets). (b) Mean number of base calls produced at various Dscore cutoffs for all SNP loci and for SNP subsets from previously published microarray platforms (NIH and Broad).
Table 1. Microarray accuracy in exons, introns, and intergenic regions
Comparative genomic hybridization performance
Segmentation analyses on multiple hybridizations of HB3 and Dd2 against the reference 3D7 detected the same copy number events detected in HB3 and Dd2 hybridizations against 3D7 found previously [25]. Median probe spacing between the 5' end of CGH probes is 52 bp, providing a fine resolution view of CNV that can precisely implicate breakpoints to within 100 bp. The resolution provided by this platform is equivalent to a previous NimbleGen CGH chip [25]. Figure 4 shows CGH scatterplots demonstrating microarray-based CNV breakpoint detection in comparison with the exact breakpoint determined through capillary sequencing.
Figure 4. CNV breakpoint detection with CGH. CGH data for a CNV on chr 5 in the Dd2 genome accurately detects the breakpoints for the (a) beginning and (b) end of the event within hundreds of base pairs. The vertical red lines indicate the breakpoint locations as previously determined through sequence data [43]. Nt, nucleotides.
CNV events are highly reproducible between replicate hybridizations with this microarray platform (Figure S2 in Additional file 1). Features, including a 500 bp CNV event, were recognized and consistent between hybridizations; however, it becomes more difficult to confidently detect small CNV events algorithmically. CNV event detection is still possible with whole genome amplification (WGA) samples (Figure S3 in Additional file 1), although the amplification process introduces noise, confounding CNV detection by any platform, particularly reducing confidence in small events. Degraded samples can not be recovered for effective CGH by WGA.
Applications to P. falciparum field samples
Standard probe labeling protocols utilize random nonamers with balanced base composition. However, P. falciparum has an extremely high AT content of 81% [28], which reduces the performance of 50% AT random nonamers and may introduce bias during amplification of the parasite genome. To test the effect of random nonamer AT composition on labeling performance and microarray data, labeling yields of 65% AT random nonamers (38,357 ± 3468.1 ng) were compared with labeling yields of 50% AT random nonamers (18,865 ± 4530.7 ng). The data pass a D'Agostino-Pearson normality test and a paired t-test, indicating that 65% AT random nonamers generate a significantly greater yield of labeled DNA than 50% AT random nonamers (P < 0.01). We see no adverse effects on base calling accuracy or CGH performance when using this modified labeling procedure.
Larger yields of labeled DNA are generated by 65% AT random nonamers, allowing the amount of initial starting DNA to be reduced. We evaluated decreasing amounts of starting material that could generate the necessary 10 μg of labeled DNA for hybridization. Labeling yields using 250 ng, 375 ng, 500 ng, and 1,000 ng of starting DNA for 3D7, HB3, and Dd2 were quantified (Figure 5a) and hybridized on the microarray. More than 10 μg of labeled DNA was obtained from all labeling reactions and no base calling or CNV detection differences were seen between the different starting amounts, indicating a starting DNA amount ≤250 ng is sufficient to generate high quality hybridization data.
Figure 5. Field sample analysis with the CNV-SNP array. (a) The manufacturer-recommended starting amount is 1,000 ng of DNA to produce at least 10 μg of labeled product. However, 250 ng of parasite DNA consistently produced sufficient labeled product when using 65% AT nonamers. Error bars indicate one standard deviation. (b) Hybridizations with field samples - straight from patient blood, or whole genome amplified - produced microarray data on par with standard lab clones, even when significant human DNA contamination was present. Microarray accuracy was determined through Illumina sequencing of lab-adapted parasites. Patient blood samples were hybridized with the addition of 1× Denhardt's solution while WGA samples were not.
Human DNA is invariably present in field-collected samples of parasite DNA and is especially high when leukocyte depletion is not used in the extraction method. In some cases, human DNA may constitute >90% of the total DNA extracted from infected blood samples and can hinder downstream uses of the parasite DNA for microarray hybridizations or sequencing. Nucleic acid blockers are commonly used in microarray hybridizations to prevent random probe binding to non-target nucleic acids and were tested on our microarray to prevent performance reduction in samples with significant amounts of human DNA contamination. We tested various nucleic acid blockers and found that 1× Denhardt's solution provided the greatest number of base calls with no negative impact on CGH data when compared to hybridizations with bovine serum albumin, dimethyl sulfoxide, human Cot1 DNA, salmon sperm, and yeast tRNA. To test for the effect of human DNA contamination on our microarray, several DNA samples were extracted from Thailand-Burma patient blood samples or were whole genome amplified from patient blood. The amount of human DNA present in each sample was quantified, and 33 to 92% of the total DNA was found to be human, with WGA samples containing the most human DNA. WGA is generally, but not always, helpful as there is some variability in data generated from WGA samples. Using 250 ng of parasite DNA, field samples were hybridized to the microarray and examined for the number and accuracy of base calls (Figure 5b); accuracy was determined using Illumina sequences generated from the same samples (M Manske, unpublished observations). This platform is able to produce high quality data with samples containing extensive host DNA contamination equivalent to data from purified lab line DNA (Figure 5b).
The CNV-SNP array provides robust, accurate data for both laboratory- and field-derived samples. Through optimizations described here, the CNV-SNP array overcomes many hurdles associated with molecular work on P. falciparum field samples. Lower starting amounts of DNA are possible when using 65% AT random nonamers that compensate for the extreme AT bias of the genome. This optimization is especially useful for field sample DNA, which is typically scarce and difficult to obtain. It also eliminates the need for in vitro culture adaptation of field samples, which is typically used to generate enough DNA for applications like next-generation sequencing and is known to alter CNV and skew results of CNV analyses [29,30]. Using our modified protocol, the CNV-SNP array requires no more than 250 ng of starting parasite DNA with no compromise in data quality. Moreover, the ample yields of labeled DNA from 250 ng of starting parasite DNA indicate that the lower limit has not yet been defined, raising the possibility that finger prick blood samples on filter paper are accessible to this technology. In addition, the CNV-SNP array is robust to samples with high host DNA contamination (>90%) with no drop in data quality, making microarray-based genotyping complementary to higher resolution next-generation sequencing that is sensitive to human DNA contamination in field samples, often requiring sample preprocessing for target DNA enrichment. Notably, high human DNA contamination and low amounts of parasite DNA present serious challenges to genotyping the large number of samples necessary for genome-wide association studies.
Probe design optimizations contribute to the performance of this microarray for the P. falciparum genome. Sense and antisense resequencing probe quartets were used for SNP genotyping on the CNV-SNP array. A SNP call required that sense and antisense probe quartets made complementary calls; furthermore, the robustness of the base call was evaluated using the ratio of background signal versus the probe with the greatest signal intensity. Signal intensities within SNP probe quartets were more similar to each other than to probes in other probe quartets or between sense and antisense probe quartets of the same locus (Figure 1) and indicates the importance of measuring the background signal for each individual SNP quartet - as provided by the resequencing probesets - rather than background noise from the entire array or locus.
Resequencing probes were optimized for SNP genotyping in P. falciparum by comparing the performance of probes at static lengths with probes balanced by melting temperature on a prototype 5K SNP array. Probe melting temperature outperformed static probe lengths for optimal SNP detection at a probe melting temperature of 66°C with performance that was reasonably consistent in exons, introns, and intergenic regions (Figure S1 in Additional file 1).
Our results on optimal probe length and melting temperature differ from findings in another study [31]. This is likely due to the use of different methods for calculating probe melting temperature and our optimization to the AT-rich P. falciparum genome. However, our broader conclusion that variable length or isothermal probes provide optimal SNP detection is supported across various organisms [31,32], and indicate that longer, isothermal probes increase signal strength while also being short enough to remain sensitive to single base mismatches [32-35].
Resequencing probesets designed for a 66°C melting temperature were generated for 45,524 SNP loci for inclusion on the CNV-SNP array. While longer, isothermal probes improve SNP genotyping, certain loci are more easily genotyped than others, and some remain inaccessible to microarrays and short-read next-generation sequencing technologies. For instance, SNPs in exons have greater genotyping success than SNPs in introns or intergenic regions, likely due to regions of high AT richness or interspersed sequence repetitiveness that hinder probe design and binding specificity in intronic and intergenic regions. Current SNP genotyping microarrays, such as those developed by the NIH and the Broad Institute [21,22], are focused on high quality SNP loci that are easily genotyped across microarray platforms (Figure 3). However, the use of isothermal probes designed at an optimal melting temperature allows us to interrogate more difficult loci and maximize the overall number of SNPs that can be robustly genotyped on the CNV-SNP array (on average, 36,948 useable SNP genotypes with 95% accuracy from a single hybridization).
An interesting debate surrounds the continued value of microarrays with the emergence of next-generation sequencing. As the cost of next-generation sequencing continues to decrease and protocols continue to improve, we will see a realization of the platform providing ultimate resolution and throughput, provoking the prediction that microarrays will soon be rendered obsolete. However, we suggest that the CNV-SNP array will continue to be useful as an 'everylab' tool alongside next-generation sequencing. Whole genome sequencing underpins the SNP discovery needed for chip design; in general, whole genome architecture and ultra-resolution mapping require fully sequenced and assembled genomes. The customizable microarray platform continues to improve in density (4.2 million element custom designs are anticipated in 2011) and offers unique configurations up to 12-plex of 135K probes, leading to a scenario in which a global set of SNPs identified by sequencing can be precisely represented on microarrays for regionally focused or hypothesis-driven designs. To date, microarrays remain cheaper, produce data more quickly, require less computational innovation, and are especially useful for processing large numbers of samples, while producing sufficient resolution and quality for genome-wide association studies and population genomic analysis. Furthermore, although progress is being made in scoring CNV using next-generation sequencing, that technology still lags behind the performance of microarray CGH.
As P. falciparum continues to evolve and evade control and eradication efforts, high-throughput, cost-effective methods of monitoring genomic variation are critical to understanding parasite adaptation. The high AT content of the P. falciparum genome is technically challenging for most molecular methods; however, the flexibility of the microarray platform described here allows users to customize and optimize microarrays to individual genomes through alterations of probe lengths, types, and numbers and adjustment of hybridization strategy. This process is applicable to population genomic studies in a wide range of organisms. Utilizing this flexibility, we created a custom high-density CNV-SNP array containing both resequencing probes capable of SNP genotyping and CGH probes for CNV detection. The CNV-SNP array is a reliable, accurate platform that allows simultaneous investigation of CNV and SNPs in a single hybridization. Its low cost, quick turn-around time, low DNA requirements, and resilience to human DNA contamination make it a valuable tool for population genomic studies.
Materials and methods
Microarray design
Probe length optimization
As an initial step in SNP genotyping optimization, a NimbleGen resequencing microarray consisting of variable length resequencing probesets was developed to assay 5,347 SNP loci. A NimbleGen resequencing probeset is composed of eight probes per SNP locus: four probes each for interrogation of sense and antisense strands. Each probe quartet is identical except for the central nucleotide that assays the nucleotide variant [27]. We downloaded 101,581 candidate SNP loci from PlasmoDB v5.5 [26] for parasite isolates HB3, Dd2, V1/S, 7G8, D10, FCC-2, K1, RO-33, D6, GHANA1, FCB, and IT. Common SNPs (SNPs identified in at least two parasite isolates) were blasted against the 3D7 genome to verify a unique 21-mer SNP typing probe sequence. Probesets with more than one exact match in the genome were discarded. Of the remaining candidate probesets, 5,347 SNP loci were randomly chosen for inclusion on the 5K SNP chip at nine different probe lengths: 21, 25, 29, 33, 35, 37, 39, 41, and 45-mers. Hybridizations following NimbleGen standard CGH procedures [36] were performed using DNA from laboratory clones 3D7, HB3, Dd2, 7G8, and D10.
CNV-SNP array
Using NimbleGen's 3-plex custom chip layout, each plex in our 3-plex 720K NimbleGen microarray contains resequencing probes for SNP genotyping and CGH probes for CNV detection (Figure 1). Probes were synthesized using maskless photolithography [37,38] with CGH probes attached by 5T linkers and resequencing probes attached with 15T linkers. Of the 101,581 candidate SNP loci downloaded from PlasmoDB and BLASTed for uniqueness, probesets for all SNPs reported in at least two parasites lines were included in the microarray. Some SNPs represented by a single isolate were included on the array and prioritized by mutation types sensitive to array detection [25]. In total, 45,524 SNP loci queried by 364,192 probes balanced to 66°C melting temperature were included on the microarray. CGH microarray probes were designed using standard NimbleGen CGH protocol [36] modified for the P. falciparum genome. Briefly, probes were tiled through the genome at 4-bp interval spacing and filtered for 60 to 80°C melting temperature and 50 to 75 bp length. The resulting probes were clustered with nearest neighbors and sorted to remove probes with extensive sequence identity to any other probe. Probes with more than one 50-mer exact match in the genome or located in hypervariable var/rif/stevor genes were discarded. The median spacing between the start of the 355,803 CGH probes is 52 bp.
Parasite samples
Fresh cultures of cloned 3D7, HB3, Dd2, SC05, and 7C126 lines derived from genotype-confirmed stock material were grown under standard cultivation conditions [39,40]. Parasite DNA was extracted using standard phenol/chloroform extraction and concentrated by salt precipitation [39]. Parasite DNA from the Thailand-Burma border was collected from 5 ml whole blood of symptomatic patients visiting malaria clinics. Buffy coats from the blood samples were removed and infected red blood cells cultured for 24 h to allow ring stage parasites to mature to schizonts to provide more DNA. DNA was extracted using a standard phenol/chloroform protocol. Parasite isolates were screened for multiple infections and identical clones using seven polymorphic microsatellite markers: ARA2 (chr 11), POLYα (chr 4), TA1 (chr 6), C2M1 (chr 2), C3M54 (chr 3), TA60 (chr 13), and C4M30 (chr 4). These markers were amplified using fluorescent end-labeled oligos and run on an ABI 3100 capillary sequencer (Applied Biosystems Inc., Foster City, CA, USA) and alleles scored using GeneScan (Applied Biosystems Inc.) and Genotyper (Applied Biosystems Inc.) software. Samples were considered multiple clone infections if one or more of the seven microsatellite loci showed multiple alleles. Only unique genotypes were included in the study. Thailand-Burma samples with an inadequate amount of DNA were whole genome amplified using phi29 DNA polymerase (Fidelity Systems, Gaithersburg, MD, USA). WGA samples were cleaned using a standard phenol/chloroform protocol. Parasite DNA concentrations in the Thailand-Burma samples were measured using quantitative PCR. Patient samples were amplified using SYBRgreen (Applied Biosystems Inc.) with primers specific to the P. falciparum ama1 gene. Reactions were run on an ABI PRISM 7900HT realtime PCR machine and DNA amounts calculated by comparison with a dilution series of pure DNA from parasite line 3D7. DNA from lab-adapted samples was submitted to the Wellcome Trust Sanger Institute (Hinxton, UK) for Illumina sequencing using 76-bp paired-end reads.
Microarray hybridizations
Labeling and hybridization were conducted using standard NimbleGen CGH procedures [36]. gDNA (250 ng to 1 μg) was denatured at 98°C for 10 minutes in the presence of 1 OD of cy3 or cy5-labeled random nonamers at 50% AT richness or 65% AT richness (TriLink Biotechnologies, San Diego, CA, USA). The denatured sample was quick chilled on ice and incubated with 50 units of Klenow fragment (New England Biolabs, Ipswich, MA, USA) and dNTP mix (6 mM each in TE (Sigma Aldrich, St Louis, MO, USA)) for 2 h at 37°C. Reactions were terminated with 0.5 M EDTA and precipitated with 5 M NaCl in isopropanol. Labeled product was resuspended in water, and 10 μg of test and reference samples combined (6 μg for 5K SNP chip samples), dried down, and resuspended in hybridization buffer (Roche NimbleGen, Inc., Madison, WI, USA); hybridizations for patient blood samples included 1× Denhardt's solution (Sigma Aldrich) in the hybridization buffer. The combined sample was denatured at 95°C for 5 minutes and allowed to hybridize on the array for 24 h (16 h for 5K SNP chip samples) at 42°C in a NimbleGen hybridization system (Roche NimbleGen, Inc.). Microarrays were washed sequentially in Wash Buffer I (2 minutes at room temperature), Wash Buffer II (1 minutes at room temperature), and Wash Buffer III (15 s at room temperature; Roche NimbleGen, Inc.) and dried for 1 minuets in a Microarray High-Speed Centrifuge (Arrayit Corp., Sunnyvale, CA, USA). CNV-SNP arrays were scanned at 2 μm resolution using a NimbleGen MS 200 Microarray Scanner (Roche NimbleGen, Inc.). 5K SNP chips were scanned at 5 μm resolution using a GenePix Pro 4200A Scanner (Molecular Devices, Inc., Sunnyvale, CA, USA). Microarray data are deposited at Gene Expression Omnibus, accession number [GEO:GSE28287].
Microarray data analysis
SNP analysis
Data for 3D7, HB3, Dd2, SC05, and 7C126 were extracted from scanned images and resequencing base reports generated using NimbleScan v2.5 (Roche NimbleGen, Inc.). Base calls were made on resequencing probesets when the sense and antisense probes made complementary base calls. The discrimination score (Dscore) for each probe quartet was calculated as a background corrected ratio of the signal from the second greatest intensity probe from a probe quartet divided by the greatest intensity probe using custom perl scripts (Figure 1, grey insert): (Second highest signal intensity - Background)/(Highest signal intensity - Background). Background for each probe quartet was calculated as the average of the third and fourth highest signal intensities [32]. There is some noise inherent in this base calling method, which can be mitigated by performing technical replicates or incorporating probe replicates into the chip design. For SNP genotyping probes, probeset melting temperature was calculated as previously described [36] where mean melting temperature was calculated from all probes in each probe quartet. The accuracy of the base calls made by the resequencing probes was calculated as the percentage of base calls that matched the reference genome for 3D7 or draft genome assemblies for HB3, Dd2, SC05, and 7C126 [20,28,41]. To ascertain the base called at a SNP locus in the genome assembly, resequencing probes were mapped to draft genome assemblies using blastall, requiring at least 95% of the probe length with less than four mismatched/indel positions excluding the central nucleotide. Illumina sequence data were aligned to the 3D7 reference genome with SNP-o-matic software [42] to identify SNP locations for comparison to microarray data.
Copy number variation analysis
Data for HB3 and Dd2 (n = 6 and n = 4, respectively) hybridized against 3D7 were extracted from scanned images and normalized using NimbleScan v2.5 (Roche NimbleGen, Inc.). Copy number events from the segmentation analysis in HB3 and Dd2 against the reference 3D7 were compared to known CNV in the published literature [25].
Bp: base pair; CGH: comparative genomic hybridization; chr: chromosome; CNV: copy number variation; SNP: single nucleotide polymorphism; WGA: whole genome amplification.
Authors' contributions
JCT conceived of the study, participated in its design and coordination, and drafted the manuscript. BAM participated in study design and coordination, performed microarray hybridizations, and drafted the manuscript. AT participated in microarray design and performed statistical analysis. IHC and TJA participated in study design and assisted with field samples. MM, GM, and DPK provided Illumina sequencing and data analysis. MTF conceived of the study, participated in its design and coordination, and helped to draft the manuscript.
This work was supported by the National Institutes of Health (AI072517, AI075145 to MTF; AI075145 to TJA). The molecular work at the Southwest Foundation for Biomedical Research was conducted in facilities constructed with support from the National Center for Research Resources, National Institutes of Health (RR013556). We are grateful to F Nosten and the Shoklo Malaria Research Unit for providing samples. We thank L Checkley, S Nair, S Al-Saai, and the Notre Dame Genomics Core Facility for excellent technical support.
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Carret CK, Horrocks P, Konfortov B, Winzeler E, Qureshi M, Newbold C, Ivens A: Microarray-based comparative genomic analyses of the human malaria parasite Plasmodium falciparum using Affymetrix arrays.
Mol Biochem Parasitol 2005, 144:177-186. PubMed Abstract | Publisher Full Text
Cheeseman IH, Gomez-Escobar N, Carret CK, Ivens A, Stewart LB, Tetteh KK, Conway DJ: Gene copy number variation throughout the Plasmodium falciparum genome.
BMC Genomics 2009, 10:353. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Ribacke U, Mok BW, Wirta V, Normark J, Lundeberg J, Kironde F, Egwang TG, Nilsson P, Wahlgren M: Genome wide gene amplifications and deletions in Plasmodium falciparum.
Mol Biochem Parasitol 2007, 155:33-44. PubMed Abstract | Publisher Full Text
Jeffares DC, Pain A, Berry A, Cox AV, Stalker J, Ingle CE, Thomas A, Quail MA, Siebenthall K, Uhlemann AC, Kyes S, Krishna S, Newbold C, Dermitzakis ET, Berriman M: Genome variation and evolution of the malaria parasite Plasmodium falciparum.
Nat Genet 2007, 39:120-125. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Mu J, Awadalla P, Duan J, McGee KM, Keebler J, Seydel K, McVean GA, Su XZ: Genome-wide variation and identification of vaccine targets in the Plasmodium falciparum genome.
Nat Genet 2007, 39:126-130. PubMed Abstract | Publisher Full Text
Volkman SK, Sabeti PC, DeCaprio D, Neafsey DE, Schaffner SF, Milner DA Jr, Daily JP, Sarr O, Ndiaye D, Ndir O, Mboup S, Duraisingh MT, Lukens A, Derr A, Stange-Thomann N, Waggoner S, Onofrio R, Ziaugra L, Mauceli E, Gnerre S, Jaffe DB, Zainoun J, Wiegand RC, Birren BW, Hartl DL, Galagan JE, Lander ES, Wirth DF: A genome-wide map of diversity in Plasmodium falciparum.
Nat Genet 2007, 39:113-119. PubMed Abstract | Publisher Full Text
Dharia NV, Sidhu AB, Cassera MB, Westenberger SJ, Bopp SE, Eastman RT, Plouffe D, Batalov S, Park DJ, Volkman SK, Wirth DF, Zhou Y, Fidock DA, Winzeler EA: Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum.
Genome Biol 2009, 10:R21. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Jiang H, Yi M, Mu J, Zhang L, Ivens A, Klimczak LJ, Huyen Y, Stephens RM, Su XZ: Detection of genome-wide polymorphisms in the AT-rich Plasmodium falciparum genome using a high-density microarray.
BMC Genomics 2008, 9:398. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Tan JC, Patel JJ, Tan A, Blain JC, Albert TJ, Lobo NF, Ferdig MT: Optimizing comparative genomic hybridization probes for genotyping and SNP detection in Plasmodium falciparum.
Genomics 2009, 93:543-550. PubMed Abstract | Publisher Full Text
Aurrecoechea C, Brestelli J, Brunk BP, Dommer J, Fischer S, Gajria B, Gao X, Gingle A, Grant G, Harb OS, Heiges M, Innamorato F, Iodice J, Kissinger JC, Kraemer E, Li W, Miller JA, Nayak V, Pennington C, Pinney DF, Roos DS, Ross C, Stoeckert CJ Jr, Treatman C, Wang H: PlasmoDB: a functional genomic database for malaria parasites.
Nucleic Acids Res 2009, 37:D539-543. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wong CW, Albert TJ, Vega VB, Norton JE, Cutler DJ, Richmond TA, Stanton LW, Liu ET, Miller LD: Tracking the evolution of the SARS coronavirus using high-throughput, high-density resequencing arrays.
Genome Res 2004, 14:398-405. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Neafsey DE, Schaffner SF, Volkman SK, Park D, Montgomery P, Milner DA Jr, Lukens A, Rosen D, Daniels R, Houde N, Cortese JF, Tyndall E, Gates C, Stange-Thomann N, Sarr O, Ndiaye D, Ndir O, Mboup S, Ferreira MU, Moraes Sdo L, Dash AP, Chitnis CE, Wiegand RC, Hartl DL, Birren BW, Lander ES, Sabeti PC, Wirth DF: Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence.
Genome Biol 2008, 9:R171. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Mu J, Myers RA, Jiang H, Liu S, Ricklefs S, Waisberg M, Chotivanich K, Wilairatana P, Krudsood S, White NJ, Udomsangpetch R, Cui L, Ho M, Ou F, Li H, Song J, Li G, Wang X, Seila S, Sokunthea S, Socheat D, Sturdevant DE, Porcella SF, Fairhurst RM, Wellems TE, Awadalla P, Su XZ: Plasmodium falciparum genome-wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs.
Nat Genet 2010, 42:268-271. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, Eisen JA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan MS, Nene V, Shallom SJ, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, Selengut J, Haft D, Mather MW, Vaidya AB, Martin DM, et al.: Genome sequence of the human malaria parasite Plasmodium falciparum.
Nature 2002, 419:498-511. PubMed Abstract | Publisher Full Text
Biggs BA, Kemp DJ, Brown GV: Subtelomeric chromosome deletions in field isolates of Plasmodium falciparum and their relationship to loss of cytoadherence in vitro.
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Day KP, Karamalis F, Thompson J, Barnes DA, Peterson C, Brown H, Brown GV, Kemp DJ: Genes necessary for expression of a virulence determinant and for transmission of Plasmodium falciparum are located on a 0.3-megabase region of chromosome 9.
Proc Natl Acad Sci USA 1993, 90:8292-8296. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Gresham D, Curry B, Ward A, Gordon DB, Brizuela L, Kruglyak L, Botstein D: Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes.
Proc Natl Acad Sci USA 2010, 107:1482-1487. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Bjornsson HT, Albert TJ, Ladd-Acosta CM, Green RD, Rongione MA, Middle CM, Irizarry RA, Broman KW, Feinberg AP: SNP-specific array-based allele-specific expression analysis.
Genome Res 2008, 18:771-779. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Chou CC, Chen CH, Lee TT, Peck K: Optimization of probe length and the number of probes per gene for optimal microarray analysis of gene expression.
Nucleic Acids Res 2004, 32:e99. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Letowski J, Brousseau R, Masson L: Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays.
J Microbiol Methods 2004, 57:269-278. PubMed Abstract | Publisher Full Text
Relogio A, Schwager C, Richter A, Ansorge W, Valcarcel J: Optimization of oligonucleotide-based DNA microarrays.
Nucleic Acids Res 2002, 30:e51. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Selzer RR, Richmond TA, Pofahl NJ, Green RD, Eis PS, Nair P, Brothman AR, Stallings RL: Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH.
Genes Chromosomes Cancer 2005, 44:305-319. PubMed Abstract | Publisher Full Text
Nuwaysir EF, Huang W, Albert TJ, Singh J, Nuwaysir K, Pitas A, Richmond T, Gorski T, Berg JP, Ballin J, McCormick M, Norton J, Pollock T, Sumwalt T, Butcher L, Porter D, Molla M, Hall C, Blattner F, Sussman MR, Wallace RL, Cerrina F, Green RD: Gene expression analysis using oligonucleotide arrays produced by maskless photolithography.
Genome Res 2002, 12:1749-1755. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F: Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array.
Nat Biotechnol 1999, 17:974-978. PubMed Abstract | Publisher Full Text
MR4/ATCC: Methods in Malaria Research. 5th edition. Manassas, Virginia; 2008.
Trager W, Jensen JB: Human malaria parasites in continuous culture.
Science 1976, 193:673-675. PubMed Abstract | Publisher Full Text
Samarakoon U, Regier A, Tan A, Desany BA, Collins B, Tan JC, Emrich SJ, Ferdig MT: High-throughput 454 resequencing for allele discovery and recombination mapping in Plasmodium falciparum.
BMC Genomics 2011, 12:116. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Manske HM, Kwiatkowski DP: SNP-o-matic.
Bioinformatics 2009, 25:2434-2435. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Nair S, Nash D, Sudimack D, Jaidee A, Barends M, Uhlemann AC, Krishna S, Nosten F, Anderson TJ: Recurrent gene amplification and soft selective sweeps during evolution of multidrug resistance in malaria parasites.
Mol Biol Evol 2007, 24:562-573. PubMed Abstract | Publisher Full Text


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iWeb for Musicians
iWeb for Musicians
Creating Offsite Links
Main Navigation:
Copyright © Roddy McKay - 2009 - All Rights Reserved
How to create image links for other websites to link to yours.
PNG Image
Image Link

A very important part of SEO is to get links to your website from other similar sites that potential customers may visit. These sites should be relevant to your own.
If you are an independent musician you can go to the Indie Link Exchange to sign up.
Another possibility with a wider range of website types is Link Lister.
When you get a response from a webmaster willing to exchange links, its a good idea to be able to offer them the code for an image link that will attract more attention than a boring text one.
The PNG image of the iWeb for Musicians button has been uploaded to a folder on the server. The PHOTO section of iWeb for Musicians shows how to create the PNG buttons.
The button image link uses the code shown below. The URL marked in blue is that of the target web page. The one in red is the URL to the PNG image on the server.
The iWeb for Musicians banner link is 480 x 104 px.
Any webmaster wishing to link to the landing page only needs to be emailed the code below with a suggestion of which page of their site that the link should appear on.
A good website for creating your own banners for free is Banner Fans.
Once you have finished your work of art, download the PNG for their website, upload it to your server and use the code on this page to display it.
The Cosmystic Music banner to the right was created at bannerfans.com.
Next Page
Find out how to get your website found by the Search Engines and keep the visitors coming back using Website Marketing.

subset: commoncrawl


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Cleo Brown Daulton
Services for Mrs. Daulton will be 2 p.m. Sunday, April 27, at Hunter Funeral Home. Interment will be in Rob Draper Memorial Garden in Gainesboro. Visitation will be 10 a.m. to 9 p.m. Saturday and 10 a.m. until services Sunday.
Mrs. Daulton, 89, of Lebanon, died Thursday, April 24, 2008, at St. Thomas Hospital.
She was preceded in death by her first husband, Jim Brown; a son, Richard Brown; a daughter, Betty Phillips; paretns, Newt and Mary Warren; two brothers; and three sisters.
Survivors include: her husband, Melvin Daulton of Lebanon; five children, Mable Bowman of Lebanon, James Brown of Norene, Grady Brown of Mt. Juliet, Charles Brown of Lebanon and Betty Fay Brown of Statesville; two sisters, Johnnie Hemontoler of Lebanon and Bonnie Thomason of Donelson; a brother, Odis Warren of Carthage; 18 grandchildren; and several great-grandchildren.
Pallbearers will be family and friends.
Jerry Gaitan
Services for Mr. Gaitan will be 11 a.m. Saturday, April 26 at Sellars Funeral Home with Bro. Danny Sellars officiating. Visitation will be 4-8 p.m. Friday and 10 a.m. until the service Saturday at the funeral home.
Mr. Gaitan, 60, of Lebanon, died Wednesday, April 23, 2008. He served in the US Navy for two years during Vietnam and worked for Nashville Display for 29 years.
Survivors include: daughters, Gerri Shontelle (Rick Sellars) Gaitan and Melody (Ricky Keel) Bell, both of Lebanon; mother of children, Cecilia Gaitan-Mobley of Madison; grandchildren, Jessica Link, and Shannon and Marcus Shane Gaitan; and granny, Theresa Brazzell.
Alota Frances King
Services for Mrs. King will be 2 p.m. Saturday, April 26 at Hunter Funeral Home with the Rev. James G. Williams officiating. Interment will be in Jennings Cemetery. Visitation will be 10 a.m.-9 p.m. Friday and 10 a.m. until the service Saturday at the funeral home.
Mrs. King, 88, of Statesville, died Thursday, April 24, 2008 at University Medical Center.
She was preceded in death by husband, Claude Thomas King; daughter, Mary Linda Cunningham; sons, Roy Donald Cunningham and Claude Thomas King Jr.; sons-in-law, Bob Wilee and Gary Eakes; granddaughter, Pamela Kay White; infant grandsons, Michael Lynn Tarpley and Christopher Michael Eakes; parents, George Robert and Carrie Naomi Dedman Barnett; step-father, Frank Harden; sisters, Dorothy Grice and Gladys Wright; brothers, Ed Barnett and Jimmy, Roy and Frank Harden Jr. Survivors include: daughters, Betty Jo Wilee of Lebanon, Carolyn Sue Eakes of Watertown and Deborah Lynn (Gary) Tarpley; son, Robert Dale (Cathy) King of Lebanon; daughters-in-law, Sandra King and Donna Jean, Brenda and Joan Cunningham; 29 grandchildren; 36 great-grandchildren; and three great-great-grandchildren; brother, George Clifton (Pearl) Barnett of Donelson; sister, Thelma Jean (Harold) Guy of Dacula, Ga.; and nieces and nephews.
Etta Moore Phillips Lester
Services for Mrs. Lester will be 2 p.m. Saturday, April 26 at Jennings and Ayers Chapel with the Rev. Charles McKnight officiating. Interment will be in Roselawn Memorial Gardens. Visitation will be 4-8 p.m. Friday at the funeral home.
Mrs. Lester, 89, of Milton, died Thursday, April 24, 2008 at Middle Tennessee Medical Center. She was a lifelong resident of Rutherford County, a member of Clarendon Chapter Colonial Dames XVII, 17th century; Colonel Hardy Murfree Chapter of the Daughters of the American Revolution; Crockett's Forde Seat Chapter of U.S. Daughters of 1812; Murfreesboro No. 91 United Daughters of Confederacy; volunteer with American Red Cross; a member of Bradley Creek Baptist and Sunday School teacher for 57 years; and a volunteer at the Veteran's Hospital.
She was preceded in death by husband, Marvin Dayton Lester; daughter, Sharon Martin; parents, Herbert and Lora Lannom Phillips; and brother, Hulan Phillips. Survivors include: sons, Herb (Pat) Lester of Antioch and Hugh (Barbara) Lester of Lascassas; son-in-law, Ron Martin of Murfreesboro; sisters, Erline Lane and Virginia L. Wilson, both of Murfreesboro; grandchildren, Shane (Dena) Martin of Laurel, Md., Ginger (Lewis) Atkins of Humboldt, Dana (Jon) Palmer, Amanda (John) Ward and Emily (John) Cauthen, all of Murfreesboro; great-grandchildren, Tyler, Callie, Lanna, Sadie, Emma and Gideon Atkins, Kaitlyn and Abigail Palmer, and Madison and Taylor Cauthen.


text:
Anthony Lewis set out to improve on the window cleaners he was using. As stated in his patent specifications, "The object of my present invention is to improve window-cleaners of this class; and to this end my invention consists in certain features of novelty that are particularly pointed out in the claims herein after".
Lewis's window washer was patented on September 27, 1882.
The four parts of his window cleaner are the handle; the body, made from a block of wood; and a flexible reservoir to absorb water made of India rubber, and the scraper. Lewis's invention was capable of cleaning several windows in succession because the reservoir could absorb a good amount of water. When applied, it thoroughly wets the window, loosens and dissolves the soil. The scraper is used to used to remove the dirty water.
Current window washers follow Lewis's design and can be purchased in most hardware stores just for a few dollars.
View Archive


text:
« MASCULINE DOSAGE: Levi McLean by Photographer James Demitri | Main | VOGUE TURKEY: Eniko Mihalik by Photographer Liz Collins »
You can follow this conversation by subscribing to the comment feed for this post.
As a final step before posting your comment, enter the letters and numbers you see in the image below. This prevents automated programs from posting comments.
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text:
Photos submitted by Local 10 meteorologist Scott Padgett's family in Dallas.
From: email@example.com [mailto:firstname.lastname@example.com]
Sent: Thursday, February 11, 2010 8:26 PM
To: Padgett, Scott; firstname.lastname@example.com; firstname.lastname@example.com
Subject: Snow Pictures
First round of pictures
This transmission may contain information that is privileged, confidential and exempt from disclosure under applicable law.
If you receive this transmission in error, do not read, use or copy it.
Please immediately contact the sender and destroy the material in its entirety, whether in electronic or hard copy format. Thank you.


text:
You are streaming Lory Del Santo - W la Foca porn video from Xhamster sex channel. Free Lory Del Santo - W la Foca sex movie was added from AmateurStore together with more porno videos. Find more celebrities, stockings, tits porn on our biggest collection of free sex videos. Well organized porno movies are updated daily.
986 views


text:
jGuru Forums
Posted By:
Posted On:
Saturday, August 23, 2003 08:02 AM
I am facing problem in having a correct display in the second tab pane of JTabbedPane. I am using JTabbedPane for displaying one panel in 1st tab and other panel (identical to 1st panel) in the 2nd tab. The panels which are added in the tabbed pane are complex panels -- having multiple panels in it with combobox, textfields, etc.
The display in the tab 1 is perfect. But when one moves to tab 2, few combobox are displayed, whereas labels, textfields are missing. The reason behind this display err is not clear. If I try to select an item in the displayed combobox, the drop down list appears & on selection of one of the item, the combobox is not displayed in the tab. So, the tab almost becomes empty. Though clicking in the area where the combobox & textfield should have been (probably), it shows drop down list & textfield part. Sometimes, few of the components come on the gui display but are again resetted(gets hidden) on some operation.
Its not the case that the panels that is being added in tab2 is having few components. If I move this panel to tab1, it is displayed correctly where as the panel which was previous on tab1 n currently on tab2, shows the unpredictible display behaviour.
Any suggestions would be very helpful.
Re: Unpredictible Display behaviour - JTabbedPane
Posted By:
Posted On:
Wednesday, August 27, 2003 11:12 PM


text:
|Curated Information Page
|PubMed Id: 9832503
|Diehl JA, Cheng M, Roussel MF, Sherr CJ (1998) Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12, 3499-511 9832503
|This page summarizes selected information from the record referenced above and curated into PhosphoSitePlus®, a comprehensive online resource for the study of protein post-translational modifications (NAR, 2012,40:D261-70). To learn more about the scope of PhosphoSitePlus®, click here.
|Information from this record has been curated, but not yet edited in PhosphoSitePlus® and may be incomplete.
|T286-p - CCND1 (mouse)


text:
Erika Freund, left, and Mama Hanz, one of the Mama Machumba women, collect banana leaves in the Tanzanian village of Meru this past summer.
- Filed Under
Erika Freund knows how to carry bananas on her head and say "you're as cool as a banana" in Swahili.
"It goes something like this: poa kichizi kama ndizi," she writes in a June 18 blog entry (http://mikutiproject.blogspot.com). "I've decided that I dig this phrase."
And while volunteering over the summer in the Tanzanian village of Meru - where overabundant banana trees provide canopies of shade - the 30-year-old graduate student learned how a simple banana tree leaf could be used to better the lives of the village women. ...


text:
- Filed Under
A pair of local charities are hitting the home stretch with fund-raising campaigns and are looking for a little more help meet their goals.
Charities in some communities are concerned about donor fatigue that has occurred with a seemingly constant stream of appeals.
Officials with the Fond du Lac Salvation Army and United Way believe there is some donor fatigue, but note that local agencies work well together to make donations stretch as far as possible.Salvation Army ...


text:
Jim Beall brings a lifetime of experience and understanding in government efficiency, transportation, and human services to the State Senate.
In three decades of public service – first as a San Jose City Councilman, then as a Santa Clara County Supervisor, and an Assembly member -- Jim Beall has left his mark across Silicon Valley.
He spurred the construction of Highways 85 and 87; fought to bring BART to San Jose; and authored bills to ease financing for seismic upgrades for our hospitals and also to grow California's solar industry. This has meant thousands of good jobs for working families.
He is known throughout California for his legislation to help foster care children, low-income families, and people with disabilities.
And he has made a lasting difference in the lives of over 100,000 local youngsters by leading the drive to create the Children's Health Initiative to ensure that every child in Santa Clara County can be covered by health insurance.
Beall plans to continue to help the people of Silicon Valley for years to come.
To read more, click here.


text:
The Central Wisconsin Racing Expo slated for February 12, 2011 at the Sunnyview Expo Center (Oshkosh Speedzone) in Oshkosh is growing on a daily basis. There will be new and used parts, tools, cars, trailers inside and outside spaces and much much more. Dan Thompson of GSR stated that if this keeps growing the way it is, this could be one of the largest racing expos in this area, something you don't want to miss. The Sunnyview Expo Centers main hall is over 25,000 square feet. If we need more space, there is more area that can be added if needed.
There will also be seminars for shocks, engines, race team sponsorships and more.
The general admission for the expo is $3.00, 11 and older.
Here is a list of vender's committed so far plus the many used vender's that are not listed. We will try to up date this when we can.
Jonny's Race Parts
Bassett Wheel
Out Pace
Wing Kart Parts LLC
TrackSide Kart / Fastrack Rep
RightFoot Performance
Mirsberger Manufacturing, LLC (parker Trailers)
Rander Car
Advantage Sign
Magnum Motors
State Wide Custom Powder Coating
E-z Bend Mid American Tool
Schaffers Oil
Snap-on Tools
Port City
Pro Graphics
Full Throttle Magazine
CFR Engines
Performance Motorsports
Stock Car Stats Pro
141 Speedway
Beaver Dam Raceway
Luxemburg Raceway
Oshkosh Speedzone Raceway
Seymour Raceway
Shawano Speedway
Sturgeon Bay Raceway
This article was posted to the website on January 24th, 2011


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The one project that started it all.
Hailed as "… the next evolution in television" by Damon Berger and a "Gigantic call to action" by Barry Jossen of ABC Television/Stage 9, Manifesto broke ground in myriad ways.
Conceived in 2005, shot later that year, and shopped over much of the northern hemisphere, the series has inspired (directly or indirectly) countless other projects. These include projects such as: The Ten Year Campaign, ABC's You Should Know, Adobe's Youth Voices, and American Express' Passion Project.
It's true, we did this all before CNN's Heroes project, too.
The series has profiled stories on the leaders of the Pay it Forward movement, Charity Water, and Julia Butterfly Hill amongst others. Manifesto continues to shoot whenever the stars align.
The son of Irish potato farmers, Scott was born in a freshly sown field during a blazing full moon in the early morning hours of the fourth of July (The year of our lord is being researched by freelance anthropologists).
Homeschooled by the family's only literate neighbor, a fireman and "clothes-free" advocate, he ran away as soon as his young legs would carry him (during the potato blight of '89) and performed as "BB" (Bullwinkle Barishnikov), a traveling sideshow where he attracted audiences with promises of 'free whiskey tasting' and then abused each of their five senses with his interpretation of modern ballet. These controversial "dance" routines are rumored to have been seasoned liberally with moshpit pirouettes.
Interviews reveal that SR believed that the Rocky and Bullwinkle children's cartoon was based on the life of an actual Russian moose who spoke English in a condescending manner.
Audiences soon grew angry once Scott championed prohibition in the shire (he was actually just out of whiskey and silver). But, due to his controversial dance performances and constant stock of taters, he was trailed by hungry talent agents across the countryside. Always fiercely independent, he hand-picked his own agent, hiring Saugus McMoughin to serve as his representation in all territories.
With Mr. McMoughin, Scott recorded his first album Tastes Like Yesterday which went on national radio and rose to #5 on the international electro-polka charts. Always reinventing his mad dulcimer-specked-harpsichord-freestyling, he released the heavy metal/acid jazz anthem Gnaw My Knickers to critical acclaim, but public uproar. Shortly after it hit the airwaves and the successive uncensored version of the "music" "video" was released, he was deported to the United States on public indecency charges.
Shunned by his home country, Scott boarded the US bound S.S. Andalusia in defeat. He now divides his time between creating epic cinematic television series for tiny interweb audiences, writing anti-Irish propaganda, and his cutting-edge tofu recycling business.
Tell me what's on your mind. I'm a good listener who enjoys a nice chat.
© 2015 Scott Ryan Online. All rights reserved.
Developed by TeoThemes


text:
|Fetal mortality in sibships of cases with neural tube defects.
MedLine Citation:
|PMID: 6388917 Owner: NLM Status: MEDLINE
|It has been suggested that rates of fetal mortality in sibships of probands with a malformation inherited as a multifactorial threshold trait may reflect their liability to the malformation. If so, spontaneous abortion rates should be more frequent in sibships thought to have greater liability. For anencephaly and spina bifida (ASB), then, spontaneous abortion should be higher in the sibships of male probands and in families with more than one affected case (multiplex families). This hypothesis was tested using data on cases from The Montréal Children's Hospital and from the literature. Approximately 5000 pregnancies were analyzed. Rates of abortion did not vary with the sex or diagnosis of the proband. The spontaneous abortion rate was slightly higher in multiplex than in simplex families, but the difference was not statistically significant and most likely reflects the differing reproductive patterns in the two types of families. Thus, if male probands and multiplex sibships do have, on average, more liability for ASB, this liability cannot be detected in spontaneous abortion rates in the sibships available for this analysis.
|A Lippman
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Publication Detail:
|Type: Journal Article; Research Support, Non-U.S. Gov't
Journal Detail:
|Title: Clinical genetics Volume: 26 ISSN: 0009-9163 ISO Abbreviation: Clin. Genet. Publication Date: 1984 Dec
Date Detail:
|Created Date: 1985-01-22 Completed Date: 1985-01-22 Revised Date: 2006-11-15
Medline Journal Info:
|Nlm Unique ID: 0253664 Medline TA: Clin Genet Country: DENMARK
Other Details:
|Languages: eng Pagination: 563-8 Citation Subset: IM
Export Citation:
|APA/MLA Format Download EndNote Download BibTex
|MeSH Terms
Abortion, Spontaneous
Anencephaly / genetics*, mortality
Fetal Death*
Spina Bifida Occulta / genetics*, mortality
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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text:
Shot in Bali on a recent break. No ND faders, no tripod and just a few lenses. No separate audio recorder, no Z-finder or monitor.
Just the camera, lenses, light stand and a gorilla pod.
You will see where a DSLR (particularly shooting in 720p) struggles. Down at the beach with very sharp lines and patterns at sunset it becomes moire and aliasing city!
You can follow me on Twitter @mattaljazeera
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Hmm…it looks like things are taking a while to load. Try again?


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Many businesses - such as grocers and hardware stores - operate on a cash-only basis after a hurricane because they lack electricity or phone service for normal processing of credit cards.
Get cash before the storm. Without power, automatic teller machines become inoperable - and cash-inaccessible. However, banks usually are on major feeder lines, which means they are amongst the first to receive power.
ATMs rely on transmission lines to talk to the computers where your bank account records are stored. Even though power may be restored, downed transmission lines could make cash inaccessible.


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Vision Inspired Photographer
Hana HarmanHair Stylist Female Perth, Western Australia, Australia
My Website: Just Hair by HanaMy MM URL: http://www.modelmayhem.com/hairbyhana
Mayhem # 1935096
About MeHello :)
my name is Hana, i am 19 years old and a qualified hairstylist from QLD who has just re located to Perth, WA. i begun hairdressing when i was just shy of 15. i have a passion for the modeling side of hairdressing and would love to work on my portfolio. i am highly trained in hair extensions and up-styles (eg; weddings, dances and catwalk/photoshoots). after signing off my apprenticeship early i have successfully ran "just hair by hana for 13 months now and due to moving would love to get my name out in Perth! i enjoy all genres of shoots and can be 99% sure to give you what you want :)
please ask questions; if you don't ask you won't ever know!
my personality is very easy going, bright and cheerful and always looking to make people smile. i am known for my witty sense of humor and honesty! the reason i enjoy hairdressing so much is simply the satisfaction and sense of achievement from fulfilling and exceeding my clients expectations.
email me: firstname.lastname@example.com
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Credit NotesModels:
Taaniela : MM # 1720676
Rob Harris : MM # 1651292
Larissa Jane : MM #1619549
Vision Inspired Photographer
Tijana Lilic Photographer
Ignite Imagery Photographer
Zarq Mozelle Model
The Shed Studios Photographer
LC Makeup and Styling Makeup Artist
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Abertay University has helped small and new businesses create prototypes of games and other interactive digital content with grants of up to £25,000. Development has been carried out at Abertay's prototyping studio, and elsewhere all around the UK.
Stormcloud Games, based in Dundee, has an impressive track-record; the highly experienced team has worked across a range of exciting technologies from Xbox360, PS3, Wii, DS, through to browser, iOS and Android.
With the help of the Prototype Fund Stormcloud Games developed the first in a series of mobile games, Mr Shingu's Paper Zoo. Paper Zoo is a creative game for children aged 6-12 years old where players fold origami animals to create their very own origami zoo and are tasked to care for, feed and home the origami animals. Colourful, fun and open-ended, the game allows players to explore their creativity through interaction and customisation.
Mr Shingu's Paper Zoo has received numerous industry and critical plaudits; as well as winning a TIGA Game Award for Best Casual game, the title has also been nominated for a Bafta Scotland award, listed as one of the top 50 apps of 2013 by the Guardian newspaper, and selected by Apple as an "Innovative Kids App" in China for the recent Apps Store Best of 2013 promotion.
Blazing Griffin is an independent game studio based in Edinburgh, Scotland. Founded in 2011, the company is a creative mix of established game developers, experts from other industries, and a wide variety of freelance talent.
With a strong focus in maintaining intellectual properties, the company has acquired the rights to games such as The Ship: Murder Party and Distant Star, with plans to expand and further develop them.
Along with buying and reviving IP, Blazing Griffin also develops new games across multiple platforms. The company helped expand the APB universe by developing APB Retribution in conjunction with Reloaded Productions. This top down action shooter was released in December 2013 on iOS.
Blazing Griffin is currently developing Dino Tribes, a match-3 game scheduled to be released in the summer of 2014 on Windows 8 mobiles, with other major platforms to follow.
Whispering Gibbon is developing a new generation of games and multimedia apps which embrace new audiences and broaden the definition of what it is to be a gamer. Combining the quality of AAA development with the innovation and usability of mobile, they are moving beyond traditional gaming, creating surprising, alternative and engaging experiences.
Tiny Trees
Tiny Trees is a uniquely relaxing experience in which player will grow, shape and nurture virtual Bonsai trees and build their very own Zen garden. The land of Tiny Trees has become overrun with invading species which have destroyed the once powerful garden; players will help banish this evil once and for as they mastering advanced bonsai techniques and grow more powerful trees. As weird and new wonderful new trees are discovered the secrets of the garden and the mystical Panda Sensei will be revealed.
FuturLab is an award winning game studio that develops and publishes original games on mobile and console, specialising in games with thrilling ideas, delightful gameplay and a refined user experience.
With the help of Abertay University, FuturLab were able to prototype a very ambitious design and take their successful Velocity franchise to a range of leading platforms.
Quartic Llama is an award-winning independent game development studio developing fun, innovative and beautiful games. The highly creative team are currently working on original intellectual property across multiple platforms, and in collaboration with exciting partners. Forming in 2012 the young studio has been named in the Develop top 100 studio list and selected as a finalists at IndieCade International Festival of Independent Games.
With the help of the Prototype Fund, Quartic Llama are developing Dama: a visually striking turn based strategy game for upcoming platforms. Dama fuses rich narrative with strategic gameplay, and features a distinctive character collection mechanic. Combine natural elements through alchemy to create new and unique creatures, explore a range of beautiful environments, and amass your collection to compete against your friends. The studio is targeting both core and casual gamers with Dama's stylised graphics, engaging strategic gameplay and unique collection mechanic.
Get in touch to find out more: QuarticLlama.com
Find Quartic Llama on Facebook: QuarticLlama
Follow Quartic Llama on Twitter: @QuarticLlama
White Paper Games was founded in September 2011 with the aim of creating original and meaningful game play experiences for a variety of gaming platforms.
Ether One is White Paper Games flagship title and is first-person adventure game that focuses on the fragility and depth of human memories. You play as a Restorer, an individual with a lost identity who is sent into the minds of mentally ill humans to restructure their broken memories. Tasked with the restoring the mind of a client named Jean you must explore the depths of her memories in order to rebuild the fragile structures within.
An increase in studio profile as well as an international awareness of Ether One has emerged from the work carried out during project and White Paper Games are now looking at releasing Ether One through a variety of different distribution avenues.
Secret Lunch is an independent games studio based in Scotland. It was founded in 2012 by a team of Master's graduates from the University of Abertay Dundee.
Their first project, Shu, is a 2.5D platformer in which players must run, jump and glide up an ancient mountain, rescuing stranded villagers from a monstrous storm.
The team have experience developing and releasing commercial games for a variety of platforms such as Xbox Love Indie Games, mobile, browser and Windows 8 and in working with companies such as Sony London and Microsoft.
GameTheNews.net is a project by digital development and consultancy studio Auroch Digital, based at Bristol Games Hub, UK. The project is exploring how games can be developed from news stories. Auroch Digital have produced a number of games on a variety of topics from the US elections to the horse-meat scandal.
GameTheNews games can be found here on their site and on Wired and the Huffington Post. The project gained international attention for its controversial newsgame, Endgame:Syria and most recently has been getting praise for NarcoGuerra. The Guardian recently said of the project, "Game the News is one of the more interesting indie game developers working at the moment…"
The focus on games that connect to reality continues to pay dividends for Auroch Digital who are doing projects with the BBC, are helping Ndemic Creations to bring Plague Inc. to Steam with Plague Inc: Evolved and will be keynote speakers at Europe's first ever newsgame hack later this year in Germany.
Hutch is a digital games studio, based in the heart of London and focusing on the highest quality 3D casual action games for iOS and Android. Their first title was the hugely popular Smash Cops on iOS and the Prototype Fund is allowing Hutch to experiment with a novel 'two-screen' game, set in the same universe.
Based on the UK's sunny south coast, in the vibrant, creative city of Brighton, Wish Studios creates innovative games and interactive experiences for home and mobile platforms. The Wish team loves games as both players and creators, and reflects that passion in its work. Wish believes strongly in the power of teamwork, bringing together the creative disciplines involved in game development to collaborate in the on-time delivery of sparkling titles.
With the help of Abertay's Prototype Fund, Wish Studios is prototyping its own game. The Fund has given the studio the creative breathing space to do this, enabling a cutting-edge new product to be brought to life, and offering a unique opportunity for the business to grow.
You can read more about Wish Studios by following it on Twitter @wishstudio or by visiting its website at wishstudios.co.uk.
Extra Mile Studios, a Glasgow, UK based developer of digital content, was formed in 2004 by a team of game development professionals with many years' experience in the children's and casual/family entertainment sectors.
The company normally works for third parties making games around existing brands, however the team are not short of ideas of their own - and with the aid of the Abertay Prototype Fund one of those has gone from the drawing board into the hands of players, and more importantly, potential publishing partners.
Phew! follows the adventures of a plucky balloon hero in his quest to stop the evil Toy Master and his minions the Moogs eradicating Phew's only friends, the Gonks.
Gameplay is all about classic side scrolling fun and action - dodge incoming attacks from both the air and ground; avoid traps and hazards; outwit the Moogs… and save the Gonks.
Moonshine Media are the west London digital marketing agency awarded an Prototype Fund grant to develop an app to promote the film 'Spike Island', set around the legendary Stone Roses gig of 1990. The app titled 'Dance Like A Manc' teaches the player the moves to master, from the 'Resurrection', the 'Bang the Drum' and the 'King Monkey' in order to dance like a proper Stone Roses fan. Once captured, the user-filmed footage is rendered on the phone with music from the film and newly originated filters, which mimic the look and feel of the feature. The footage is then uploaded to the YouTube site as part of our competition to find those with all the best Manc moves.
In order to 'Dance Like A Manc', and other examples of the brilliant, and stylishly creative work out of the Moonshine stable, click here: http://www.moonshinemedia.co.uk/ or follow this: @welovemoonshine
Nice one!
Binary Pumpkin opened its Dundee doors in 2012 and it's first product, Pumpkin Bingo was launched at Christmas of that year. With the help of the Prototype Fund, the company was able to start employing staff and develop a prototype called TaxiTaxi. Developed using the eeGeo 3D map platform, the game puts the player in the shoes of a taxi dispatcher who needs to source the best routes to guide fares across San Francisco whilst avoiding ongoing incidents, traffic jams and other city chaos.
The prototype has received positive feedback from potential publishing partners and Binary Pumpkin are hopeful of securing the additional funding to bring the game to the market in due course.
Outplay Entertainment are a Dundee based, venture backed social and mobile game developer with several successful titles across iOS, Facebook and Android. The prototype fund has allowed Outplay to create Media Frenzy!, a media rich, online multiplayer trivia experience for tablet and mobile. Players engage in unique interactive rounds to answer questions on their favourite movies, music, TV and celebrities. Media Frenzy!'s close integration with media partners allows players to discover, engage and socialise around the media enjoyed in each round.
Specializing in motion control and optical recognition technologies, Peekabu Studios was founded in 2010 and has developed applications for the web as well as smart TVs. The Prototype Grant helped Peekabu develop a web game promoting the upcoming film "Smash and Grab," the real-life story of a gang of jewel thieves. To put players in the heart of the action, the game uses Peekabu's motion control technology to track players' hands as they listen at the safe door, turn the combination dial, and try to crack the safe and steal the loot without being caught. Players need a steady hand, steely nerve, and the will to be calm under pressure to match the thieves in the film. To hear about launch dates and other news, visit http://peekabu.net.
Lucky Frame is a small company specialising in quirky and fun games and interfaces, particularly with regards to music. Their work includes iPhone apps, desktop computer software, games, and performance systems, all of which aim to find new ways to encourage creativity through technology. Lucky Frame are working on Martin vs Monty, a two person deathmatch game for iPad with the help of the Prototype Fund. Featuring a Victorian era styling and fast paced gameplay reminiscent of 1990s arcade games the team hope to build upon the recent success of Bad Hotel, the BAFTA winning iPad game.
Euphonious is an audio production company founded in 2011 by multiple BAFTA winning Raymond Usher. The award from the Prototype Fund has allowed Euphonious to kickstart development of the next generation of audio middleware which addresses the new challenges faced by today's game developers.
Clockwork Racers is a multi-screen remix of Micro Machines and Mario Kart: wind-up your favourite Clockwork Hero (Squeeks, Zapp, Chomp or Snooze) and race through 16 miniature environments with your family and friends. Clockwork Racers connects any and all of your devices, with secret weapons available on your phone while everyone's attention is on a shared tablet, laptop or smart tv.
The Prototype Fund helped Opposable Games develop their multi-screen technology further and launch the first of the Clockwork games for the Antix and iOS games platforms. Opposable is made up of seven of the South West's best games designers and developers, and collaborates with publishers, developers and IP holders to develop connected, multi-player games on mobile, tablet, set top box, and smart TV.
Guerilla Tea is a Dundee based indie games and software developer. Founded in 2011 Guerilla Tea is primarily a service-based developer doing work for companies in a range of different industries.
The Prototype Fund has allowed Guerilla Tea to take a temporary step back from contract work to develop our own self-owned piece of IP a model helicopter dogfighting game currently named 'Fangs Out!'. The game was originally developed for mobile and table devices but we have recently turned our attention to the possibility of turning it into a PC game. To keep up with Guerilla Tea's latest news check out or website www.guerillatea.com.
After a cataclysmic global war Civilisation lies in ruins. Survivors are now just emerging from underground bunkers. It is a new era. It is the first year after the Apocalypse. It is Year 0…
Year 0 is a browser-based, multiplayer, online, strategy game. Players battle for survival and to mould the new world in their image. The Prototype was developed by Legendary Games an R&D focused games company using HTML 5 technology that will allow the game to be accessed from any browser equipped device and embedded in any browser carrying platform be it PC, Mac, smart phone, console or Kindle. Visit the company website for more information: http://legendary-games.com/
Supersonic have a long history of console development and have recently refocused their development onto mobile projects. Working with a high profile UK charity, Supersonic are prototyping a mass-market endless-runner game featuring the charity's mascot characters with the help of the Prototype Fund. The game is being developed for iOS and Android devices. The game is intended to be compelling, fun and innovative in its own right, as well as (importantly), spreading the message about the work of the charity.
PLA Studios was founded in 2011 in East London and is a primarily service-based game developer. PLA develop games for publishers, educators, brands and entertainment and have grown rapidly over the course of 2012.
The Prototype Fund grant has allowed PLA to develop a HTML5 game under own IP alongside client projects. "Squaddies", PLA's social mercenary hire 'n' fire 'em up, is being developed for the Turbulenz web gaming platform and, even though it is still in fairly early stages, you can follow its development at http://squaddies.tumblr.com
Founded in 2010, Setgo is led by three experienced industry professionals who previously worked closely together as the senior management team of Sony's Studio Liverpool. With the help of the Prototype Fund Setgo developed Castaways; a Facebook game that sees the player stranded on a mysterious island where they must survive, prosper and maybe even escape. Although we are no longer actively developing new content for it, the game itself remains live on Facebook and on our website. One of the biggest lessons learned over the course of the prototype was the importance of metrics and analytics during the development of Free-to-Play games. From that Setgo developed Pingflux, one of the first truly platform agnostic game analytics solutions in the market. Through Pingflux, developers are provided with insights into the behaviour of their players, regardless of which platforms they've chosen to adopt for their game. Pingflux works for games on smartphones, tablets, browser, social or any other platform that's connected to the internet. Check out the website at www.pingflux.com or follow Pingflux on Twitter @Pingflux.
OM is a leading producer of dialogue for games. The Prototype Fund is supporting the commercialisation of a suite of creative dialogue tools developed in-house to enhance creativity. The toolset revolutionises recording, post-production, review and QA of voice content. The core focus of the tools is in empowering directors and actors during recording to enable more subtle and connected performances. The bi-products are fast turnaround, greater accuracy, multi-level QA at source and significant cost savings.
Two Monocle Games creates social games for existing networks such as Facebook. First setting sail in 2011, their first title Cult Leader offers a satirical look at social games, politics and celebrity culture. The Prototype Fund proved vital in helping the company to create their first product in covering staffing costs. Two Monocle also took advantage of the Prototyping Studio space and equipment available at Abertay University. After the toil and adventure of Two Monocle's maiden voyage the company is now preparing to set sail for their next expedition. Who knows what awaits them in the mysterious uncharted waters of games development? Whatever happens, it will certainly be a great adventure.
WildKnowledge is a spin out company from Oxford Brookes University who specialise in data gathering and exploration mobile apps. WildKnowledge were seeking to incorporate a higher degree of gamification into their applications and used the prototype funding to create an app to raise awareness of National Geographic's Big Cat Initiative. National Geographic were keen to explore how users could access their content in an engaging way. The Big Cat Challenge enables users to become Super Rangers by visiting Big Cats and Rangers around the world and discovering facts and threats to these creatures. You can only become a Super Ranger if you seriously know your stuff! It is anticipated that the Big Cat Challenge will be launched for the iPhone by the end of the year.
Bigyama is a registered developer for PlayStation Vita and PlayStation Home. The prototype grant was used to prototype a PlayStation Home space and develop the backend server technology needed to make more involved Home games. Bigyama develops virtual goods for PlayStation Home, has brought Fallen Tree's Quell Memento game to PlayStation Vita and has been undertaking contracting work to keep everything ticking along.
Established in November 2010, The Connected Set design and produce content, TV formats, games and digital products. The Connected Set used their prototype grant to develop Tate Britain QuizTrail, a location-based iPhone game that guides players on themed trails around the famous London gallery. Each trail has three levels of play that cover simple observational questions to tougher multiple-choice questions requiring players to read display captions to find the answer. Players are rewarded with a discount at the Tate Online Shop. Visitors can share their new found knowledge of the works with friends as they progress on their trail and share their prizes too via a bespoke Facebook app. The Connected Set will be porting the game to Android later in 2012 and are already in talks to develop versions of QuizTrail for other museums and galleries in UK, Europe and the US.
Dry Ice Studios' first title will be a side-scrolling adventure distributed via digital download. Solid game play and high production quality are very important factors and we plan to keep our development team small and focused.
Triple B Games is an indie developer based in Dundee. Founded in 2007 we have released five titles on the Xbox 360's Indie Games channel: Fitba, a particularly Scottish football game; Inside Lacrosse's CL2010, the first Lacrosse game on the Xbox 360; NLL 2010, the first officially licensed Lacrosse title; College Lacrosse 2011; Zombiez 8 My Cookiez; and Flowrider. Flowrider uses a large scale realtime fluid dynamics simulation to provide a unique top down racing experience. Flowrider features online, split-screen and single player modes with futuristic visuals in full HD and a custom-composed soundtrack. Flowrider is coming soon to Xbox 360 Indie Games.
Bookmarked Games is a partnership between educational gaming entrepreneur Jeremiah Alexander of Ideonic and author & public policy strategist Oliver Roberts.
Although their first game is currently in development, we can't say too much yet. What we can say is that it will focus on stories which play a major role in early childhood development. We're one of just six companies in the UK to receive a grant from the first round of the UK's new game prototyping fund, run by the University of Abertay. That means the game concept's already been through a rigorous vetting process in its development; and that we have the backing of the University's expertise to help us make it a success.
Alongside supporting new businesses, the fund also aims to build skills in the UK's digital sector, by paying for recent graduates to help us build the game. We're fully committed to the aims of that fund, and so by taking people on we're also committed to their development, bringing on their skills and ensuring this project helps launch their careers in this sector.
14 Principles provides tools, training, and consultancy to make teams more effective. For years, running game projects has been about the wrong thing: cliff-edge releases, go big or go home, large teams, and large budgets. Our lightweight, delivery-focussed approach draws upon lean and agile practices. It's ideally suited to small game development teams looking to bring brave, creative ideas to market without betting the farm.
Future Fossil Studios is an independent games developer, currently producing their own intellectual property and engaging with the education sector to teach a younger generation about the skills behind of games development.
With the aid of IDEAScotland and the Prototype Fund, Future Fossil developed a proof of concept to address the issues surrounding technical education and are now seeking partners to further their interests in this field. The product presents an accessible, easy to use platform to teach the very basics of games development, and unleash an audiences creativity. Having undertaken work with organisations such as the Prince's Trust, Future Fossil is positioning itself at the forefront of games education.
Future Fossil was founded on the belief that games should be fun and engaging experiences, with the power to educate and shape how an audience thinks.
To find out more about Future Fossil Studios, visit them at their website, facebook, and twitter.
Combo Games is a Dundee based company with origins in Denmark. The development team at Combo Games consists of award-winning developers, local talent from Dundee and amazing freelancers contributing work for our first own-IP development, Whitespace.
Whitespace is a competitive multiplayer game taking place in a futuristic sci-fi setting. Players battle for fame and fortune in the world of Whitespace at an abandoned vacation resort, called The Last Resort, which is controlled by a schizophrenic A.I. Players control one out of many different avatars in five versus five team matches to satisfy the ruthless needs of the A.I.
Whitespace is introducing multi-perspective combat for the first time ever in the MOBA-genre, allowing players to battle with and against each other from two different perspectives. When playing from first person perspective, players will focus on precision and agility to succeed in combat, while playing from top-down perspective allows players to gain a tactical advantage through superior spacial awareness and strategic options. This duality allows for a wide range of play styles and encourages players to find the play styles that suits them.
To learn more about Whitespace and the team feel free to visit Combo Games at www.whitespacegame.com or www.facebook.com/Whitespacegame
Swallowtail Games is a company formed of Abertay University graduates and Dare to be Digital Alumni.
Abertay University's Prototype Fund helped Swallowtail Games develop the BAFTA Ones to Watch winning game Tick Tock Toys, a colourful puzzle game aimed at children and families for iPhone and iPad. The game's main character "Tic Toc" will wind his key four times, and make his way to the goal along the chalk line on the toy box. The toys in Tic Toc's way have various touch based mechanics that the player must use to prevent him from bumping into them and falling down. The player must also find Tic Toc's missing puzzle pieces by tapping on the toys. The Prototype Fund was key in helping Swallowtail Games start up, providing studio space and equipment.


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The All Out Trinity Festival is taking place on the Margaret Hunt Hill Bridge and along the western side
of the Trinity River basin beneath the bridge. Most of the access to the festival is from West Dallas,
near the Trinity Groves dining district.
Please note: some roads leading into the festival area are closed due to construction. Also, parking
will be competitive, with 3,000 athletes coming in for the Trinity River Levee Run, the Trinity Sprint Invitational and the Glenn Carter Public Safety Challenge. Once you're here though, you'll have an
epic time.
We strongly encourage festival-goers to consider biking to the festival or using a ride-share service such as Uber, Lyft or a taxi.
Biking to the Festival
Driving Directions From:
02.08.14 Trinity River Levee Run
Registered yet for our 10k, 5k or one-mile Fun Run and Walk on Mar. 1? Kick off All Out Trinity with the event that inspired the rest of the day: the Dallas Running Club's 10th Annual Trinity River Levee Run.
The Trinity Commons Foundation and City of Dallas Trinity Corridor Project thank these sponsors for their support of All Out Trinity.
Future Trinity Commons sponsorship opportunities
The Trinity Commons Foundation and City of Dallas Trinity Corridor Project also appreciate these planning partners.


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Updated 08/19/2014
The sign outside will likely contain a plug for the virtues of garlic. Rightly so, for no one in Fairbanks puts fresh chopped garlic to better use than Geraldo's, which has gourmet pizza, seafood, pasta, and veal dishes. A painting of Don Corleone hangs on the wall, and Frank Sinatra and Dean Martin provide background music for this cozy and often crowded spot.
701 College Rd., Fairbanks, Alaska, 99701, United States
Updated 08/19/2014
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Developers hope Capitol Art Lofts, across from the King Edward Hotel, will create an arts district in downtown.
- Filed Under
The development team behind the renovation of the King Edward Hotel and Standard Life buildings in downtown Jackson is turning its attention to building loft apartments nearby that could serve as an artistic hub for the metro area.
HRI Properties, based in New Orleans, hopes to start construction in February on the $10 million-$11 million Capitol Art Lofts, to be built throughout seven vacant buildings in the 200 block of West Capitol Street. The buildings, which the company has rights-to-purchase agreements on, are across from the King Edward that reopened in late 2009 after a long ...


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Sesame-Soy Broccoli Florets Recipe
5 4
"We love to eat healthy, and this recipe has an Asian flare with a touch of sweetness that's sure to satisfy even those who turn up their noses at broccoli," says Marianne Bauman of Modesto, California.
- 2 cups fresh or frozen broccoli florets
- 1 tablespoon sugar
- 1 tablespoon olive oil
- 1 tablespoon reduced-sodium soy sauce
- 2 teaspoons rice vinegar
- 2 teaspoons sesame seeds, toasted
- Place the broccoli in a steamer basket; place in a saucepan over 1 in. of water. Bring to a boil; cover and steam for 5-7 minutes or until crisp-tender.
- Meanwhile, in a small saucepan, combine the sugar, oil, soy sauce and vinegar. Cook and stir over medium heat until sugar is dissolved. Transfer the broccoli to a serving bowl. Drizzle with soy sauce mixture; sprinkle with sesame seeds. Yield: 2 servings.
Originally published as Sesame-Soy Broccoli Florets in Taste of Home April/May 2006, p59
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Reviewed Sep. 18, 2014
"I've made this numerous times over the past 5 years and it always turns out excellent. Nice flavor and easy to increase for more than 2."
Reviewed May. 24, 2014
"This was a nice side dish for a family dinner."
Reviewed Sep. 23, 2011
"This sauce is so good on top of broccoli!!"
Reviewed Sep. 19, 2009
"Best broccoli we've ever had!~ Theresa"