Recent epidemics of dengue viruses (DENV) coupled with new outbreaks on the horizon have renewed the demand for novel detection methods that have the ability to identify this viral pathogen prior to the manifestation of symptoms. The ability to detect DENV in a timely manner is essential for rapid recovery from disease symptoms. A modified lab-derived 10-23 DNAzyme tethered to gold nanoparticles provides a powerful tool for the detection of viruses, such as DENV.
We examined the effectiveness of coupling DNAzyme (DDZ) activation to the salt-induced aggregation of gold nanoparticles (AuNP) to detect dengue virus (DENV) progeny in mosquito cells. A DNAzyme was designed to recognize the 5’ cyclization sequence (5’ CS) that is conserved among all DENV, and conjugated to AuNPs. DDZ-AuNP has demonstrated the ability to detect the genomic RNA of our model dengue strain, DENV-2 NGC, isolated from infected Aedes albopictus C6/36 cells. These targeting events lead to the rapid aggregation of AuNPs, resulting in a red to clear color transition of the reaction mixes, and thus positive detection of the DENV RNA genome. The inclusion of SDS in the reaction mixture permitted the detection of DENV directly from cell culture supernatants without additional sample processing. Specificity assays demonstrated detection is DENV-specific, while sensitivity assays confirm detection at levels of 1 × 10 1 TCID50 units. These results demonstrate DDZ-AuNP effectively detects DENV genomes in a sequence specific manner and at concentrations that are practical for field use.
We have developed an effective detection assay using DNAzyme catalysis coupled with AuNP aggregation for the detection of DENV genomes in a sequence specific manner. Full development of our novel DDZ-AuNP detection method will provide a practical, rapid, and low cost alternative for the detection of DENV in mosquito cells and tissues, and possibly infected patient serum, in a matter of minutes with little to no specialized training required.
Dengue viruses (DENV), members of the Flavivirus family of viruses, cause periodic explosive epidemics in many tropical and sub-tropical countries leading to approximately 50-100 million infections per year [ 1 - 5 ]. Approximately 500,000 of these are severe cases requiring hospitalization with a 2.5% fatality rate, most of which are children [ 1 - 5 ]. Approximately half the world’s population remains at risk for DENV infection making this pathogen one of the most dangerous viruses in the world [ 6 ]. In 2010 there were 1.6 million cases of Dengue in the Americas alone, of which 49,000 were severe cases [ 1 ]. Recent domestic outbreaks have occurred in the Hawaiian Islands in 2001 [ 7 ], Brownsville, Texas in 2005 [ 8 ], the Florida Keys in 2010 [ 9 ] and other parts of southern Florida including Miami-Dade in 2011 [ 10 , 11 ]. Furthermore, devastating outbreaks continue to occur in Puerto Rico [ 10 , 12 , 13 ], Brazil [ 14 ], and Pakistan [ 15 ], to name a few.
DENV are maintained in a cycle that involves humans and the globally disseminated Aedes aegypti mosquito [ 16 ]. Infection with one of four antigenically distinct, but genetically related DENV serotypes (designated DENV-1, -2, -3, and -4) can result in dengue fever (DF) and/or potentially fatal dengue hemorrhagic fever (DHF) [ 17 ]. These disease states are characterized by high fever, often with enlargement of the liver, and in severe cases circulatory and respiratory failure [ 3 ]. While DF and DHF are endemic to tropical and subtropical regions of the world, collapse of effective vector control programs, rapid dispersal of viruses due to ease of global travel, and migration of humans from tropical to non-tropical regions has resulted in DENV outbreaks in regions that were once non-endemic to these viral pathogens.
The ability to detect DENV in a timely manner is essential to rapid recovery from disease symptoms. Currently, detection of mosquito-borne viruses in infected persons is limited to plaque assays, antigen detection assays (e.g. NS1 antigen detection), or quantitation of viral production through PCR-based methods [ 18 - 20 ]. These assays are currently referred to as the “gold standards” for DENV detection [ 21 - 24 ]. More relevant to our research, current testing of mosquito populations for arboviruses in general, but more specifically dengue viruses, has been limited to RT-PCR of mosquito pools (25-100 insects) [ 25 - 27 ].
The approaches mentioned above are limited by a number of pitfalls including low-throughput, labor-intensiveness, low stability of assay components at or above room temperature, and lack of portability. The requirement for specialized training and equipment and the time consuming nature of these assays limits their widespread utility for virus detection. These limitations compromise rapid diagnosis of viral infections. Additionally, these methods are not easily adapted to field environments where reliable and effective detection methods are needed. Rapid, low-tech virus detection methods that require no specialized training or education are sorely needed to provide remote areas of the world the ability to detect highly pathogenic viruses for both clinical diagnosis and epidemiological surveillance.
In this report we describe the development and initial validation of a colorimetric DENV detection method that couples the RNA targeting ability of a DENV-specific DNAzyme (DDZ) with the aggregation properties of oligonucleotide-tethered, noncrosslinking gold nanoparticles (AuNPs). Our innovative DENV detection system, called DDZ-AuNP (Figure 1), should be an invaluable tool for the detection of DENV since it solves many of the limitations of current virus detection assays. This assay and subsequent analysis is cost effective, simple to perform, and the assay components are highly stable at temperatures above 30°C enabling easy storage at room temperature. The use of DNAzymes in the assay increases the specificity and versatility of detection permitting the design and incorporation of additional virus or strain-specific DNAzymes and probes.
Overview of the DDZ-AuNP assay for dengue virus detection. Schematic of the DENV detection system using DENV-specific DNAzyme (DDZ) catalysis coupled with gold nanoparticle (AuNP) aggregation. AuNPs are conjugated with the sulfide-linked anti-DENV DNAzyme, DDZ, which is complimentary to the DENV RNA genome (shown in orange). Black vertical lines indicate complimentary base pairing between DDZ and the target RNA. In the presence of DENV RNA (A), the 5’ and 3’ arms of the anti-DENV DNAzyme, DDZ, bind to the 3’ and 5’ ends of the targeted 5’-3’ CS region, respectively (B). When Mg2+ and heat (37°C) are introduced DDZ digests the viral RNA (C). This digestion results in deshielding of the AuNP, leading to aggregation of these AuNPs in the presence of NaCl and heat (D) allowing a rapid and visually detectable red to clear color transition (E). This color transition signifies the successful detection of DENV, and can be quantified by UV/Vis spectrophotometry at 520 nm AuNPs = red, tethered DNA probe = orange, DENV genome = purple.
Full development of this detection assay would greatly enhance virus diagnostics and epidemiology by providing an assay that is more rapid, easier to use, has greater portability, and is more cost effective than current DENV detection methods.
The dengue virus detection method described below (see Figure 1) is based upon RNA aptazyme-mediated detection of small molecules, such as theophylline [ 28 , 29 ]. The central limitation of using RNAzymes and aptazymes for virus detection is the inherent instability these catalytic RNAs, increasing the attractiveness of DNAzymes in detection assays.
The colorimetric detection of DENV by DDZ-AuNP can be divided into three phases: targeting/cleavage of the DENV RNA genome by DDZ, activation of AuNPs, and aggregation of AuNPs and detection (Figure 1). In the presence of DENV the 5’ and 3’ arms of DDZ bind to the 3’ and 5’ ends of a fully conserved segment that includes the 5’-3’ CS region, respectively, and in the presence of Mg 2+ and heat (37°C) the viral RNA is cleaved at G149. This cleavage results in deshielding and aggregation of the AuNPs in the presence of NaCl and heat [ 30 - 32 ], causing a visually detectable red to clear color transition [ 31 , 32 ] that can be quantified by UV/Vis spectrophotometry at 520 nm [ 31 , 32 ]. If DENV are not present in the sample, the DNAzyme -tethered AuNPs remain in a dispersed state and no color loss should occur [ 31 , 32 ]. Likewise, if any of the essential components such as magnesium or sodium are not present in the reaction mixture, no aggregation is possible.
DNAzymes are lab-derived, auto catalytic DNAs consisting of three intimately connected domains (Figure 2A): A catalytic core that is activated by binding a cofactor (eg. Pb 2+ or Mg 2+) [ 33 - 36 ] (though a few DNAzymes do not require a cofactor) [ 37 ], and 5’ and 3’ binding arms that bind to the 3’ and 5’ regions of the target sequence, respectively. DNAzymes have demonstrated impressive sensitivity in detecting metal ions or RNA [ 38 - 45 ].
Anatomy of the 10-23 anti-DENV DNAzyme (DDZ) and Schematic Diagram of the DENV genome. A) DDZ was designed and produced as previously described [ 36 , 85 ] and in Materials and Methods. R = Purine. Y = Pyrimidine. The target RNA is in green with the anti DENV DNAzyme 10-23 shown in black. The 5’ and 3’ ends of target RNA and DNAzymes are as indicated. Thin black vertical lines show complimentary base pairing. Arrows indicate cleavage site of the target RNA. B) A representation of the 10,723 base DENV-2 NGC GC capped and polyadenylated genome is shown to illustrate the position of the region targeted by the anti-DENV DNAzyme, DDZ. Non structural (NS) genes 1 through 5 are shown in green. Structural genes encoding capsid, membrane glycoprotein precursor, and envelope proteins are shown in red. UTR = untranslated region, PA = polyadenylation.
The 10-23 DNAzyme is capable of cleaving RNA with high sequence specificity at target sites containing purine-pyrimidine (R-Y) junctions [ 46 ]. We chose the 10-23 DNAzyme for use in our DENV detection system because this DNAzyme is less dependent on secondary structure formation for its activity than other DNAzymes [ 47 ] and would be expected to perform better in our in vitro assays where biomolecular folding would be quite variable. The design of the anti-DENV 10-23 DNAzyme, DDZ-M (Figure 2A), was based on a 10-23 DNAzyme clone that was discovered through SELEX (Systematic Evolution of Ligands by Exponential Enrichment). We designed the 5’ and 3’ arms to target the highly conserved region that includes the 5’-3’ cyclization sequence (CS) that is present in all DENV serotypes, and is required for replication of genomic RNA (Figure 2A) [ 48 , 49 ].
Gold nanoparticles (AuNPs) ranging from 15 nm to 100 nm in diameter have been used in a number of detection assays [ 30 , 50 , 51 ]. We chose to conjugate DDZ to 15 nm AuNPs since fewer copies of single-stranded DNA are required to cover the surface of a 15 nm AuNP than any AuNP of larger size [ 52 ], and interaction of only 7.5% of DNAs conjugated to the 15 nm AuNPs with the substrate RNA is required to initiate aggregation of the AuNPs [ 30 ].
AuNP-conjugated DDZs were analyzed for their ability to cleave the DENV-2 NGC RNA in vitro. DENV-2 NGC viral RNAs were isolated from infected Ae. albopictus C6/36 cells, and incubated in a buffered solution containing 1 × 10 5 DDZ-M-tethered AuNPs/mL for 30 minutes at 37°C. Digestion products were then amplified by RT-PCR using heterologous and hexamer primers designed to aid in the amplification of DDZ digestion products.
Successful digestion of the DENV-2 NGC RNA genome by DDZ-M was demonstrated by the positive detection of 2 fragments of approximately 150 and 350 bases in size, indicative of DDZ-M catalysis (Figure 3). DNAzyme catalytic activity against the DENV-2 RNA genome was validated by the inclusion of an inactive DNAzyme negative control, DDZin-M, that was created by inverting the catalytic domain which has been previously shown to render the DNAzyme catalytically inactive [ 41 ]. As expected, the catalytically inactive DDZin-M did not digest the DENV-2 NGC genome due to this alteration in the catalytic domain.
In vitro DDZ activity assay. DDZ-M-AuNPs (1 × 10 5 particles/mL) were placed in a buffered solution containing 10 mM MgCl
As an initial test of the utility of our colorimetric detection method a synthetic target was designed and synthesized that corresponds to the 5’ 170 bases of the DENV-2 NGC genome. This stretch of nucleotides included the highly conserved 5’ CS domain and the initial 74 bases of the capsid gene [ 49 , 53 , 54 ].
Synthetic target (7.5nM) was added to a buffered mixture containing 1 × 10 5 DDZ-AuNPs/mL, 10 mM MgCl
Colorimetric DDZ-AuNP detection of a synthetic DENV-2 NGC RNA target. A synthetic stretch of ribonucleotides corresponding to the 5’ 170 bases of the DENV-2 RNA genome was added to a buffered mixture containing 10 mM MgCl
Sodium, in the form of NaCl, is an essential component of AuNP colorimetric detection assays because this monovalent salt drives aggregation of oligonucleotide-conjugated AuNPs [ 30 , 55 - 57 ] following the interaction of the AuNP conjugated probes with complimentary oligonucleotide targets [ 58 , 59 ]. However, NaCl concentrations greater than 2 M can cause instability of conjugated AuNPs [ 30 ]. Furthermore, published reports indicate that NaCl concentrations for effective AuNP aggregation can vary from 1.0 M to 1.5 M [ 51 , 60 ]. In light of these prior observations, we evaluated the optimal NaCl concentration necessary to initiate aggregation of DDZ-M-AuNP following interaction with the DENV-2 genome.
DENV genomic RNAs (~0.6 μM), isolated from infected C6/36 cell supernatants, were added to a buffered reaction mixture containing DDZ-M–AuNP (~1 × 10 5 particles/mL), 10 mM MgCl
Determination of optimal NaCl and SDS concentrations. A) The optimal concentration of sodium, in the form of NaCl, for aggregation of DDZ-M-AuNP following interaction with the DENV-2 RNA genome was determined. DENV -2 NGC strain genomic RNAs were isolated as described in Materials and Methods and 0.6 μM was incubated in a reaction mix containing DDZ-M-AuNP (1 × 10 5 particles/mL), 10 mM MgCl
Our DDZ-AuNP assay system demonstrated utility in detecting purified DENV-2 RNAs. However, to improve this assay for field use we needed a protocol that has speed, efficacy, and simplicity in detecting DENV RNA directly from virions. Liberating the DENV RNA genome from virion particles using a low cost, non-toxic RNA extraction reagent that is stable in the reaction buffer and does not interfere with the assay would be ideal. Sodium dodecyl sulfate (SDS) is an effective non-ionic detergent for lysing virus particles [ 62 ]. SDS may be considered an ideal component for our colorimetric detection assays because it is non-toxic, stable in the reaction buffer, and does not require additional manipulation during lysis.
The optimal concentration of SDS was determined by adding cellular supernatants containing 1 × 10 6 DENV-2/mL to buffered reaction mixes containing DDZ -tethered AuNPs (DDZ-AuNP), 10 mM MgCl
Since DDZ is activated by 10 mM MgCl
Assessing Mg 2+ resistance of DDZ-tethered AuNPs. DDZ-M tethered AuNPs were incubated with increasing concentrations of MgCl
Because Chikungunya virus (CHIKV) and DENV co-infections have become more prevalent in South Asia and Africa [ 63 - 65 ], we tested our DDZ-AuNP detection method for its specificity for DENV in the presence of CHIKV (Figure 7A). Cellular supernatants containing 1 × 10 6 DENV-2 or 1 × 10 6 CHIKV/mL, as determined by TCID
Assessment of DDZ-AuNP specificity. A) DENV-2 and CHIKV (1 × 106/mL each) were placed in a buffered solution containing 10 mM MgCl2, 1 × 105 DDZ-AuNP particles/mL, 1.5 M NaCl, and 0.5% (w/v) SDS. Eppendorf tubes containing these mixes were incubated at 37°C for 30 minutes and photographed. CHIKV = chikungunya virus. DENV-2 = dengue virus serotype 2, DDZ-M = anti-dengue virus DNAzyme, DDZin-M = inactive anti-dengue virus DNAzyme. B) Analysis of DENV detection by UV/Vis Spectrophotometry. Samples were assembled as was performed for in A, mixed, incubated at 37°C for 5 minutes, and spectrophotometric analysis was performed using the ND-1000 spectrophotometer. C) Detection of DENV by DDZ-M-AuNP in comparison to several other flaviviruses. The specificity of our DDZ-M-AuNP device to detect DENV and not other fellow flavivirus members YFV, JEV or ZV was determined as described earlier (see Figure 4). D) An alignment was performed on consensus sequences of each of the four DENV serotypes to determine the most optimal regions for the design of serotype specific DDZ-AuNP devices by determining the region of least conservation one serotype has with the other DENV serotypes. E) Colorimetric serotype-specific detection of DENV. Cell culture supernatants from C6/36 cells mock infected (Mock), or from cells infected with either DENV serotypes 1 through 4 or CHIKV (1 × 106/mL each) were placed in buffered solutions containing the necessary cofactors and AuNPs tethered with our all purpose DENV serotype specific DNAzyme, DDZ-M, or one of the serotype-specific DDZs (designated DDZ-1 through-4) designed to specifically target the corresponding DENV serotype (Table 1 ). Eppendorf tubes containing these mixes were incubated at 37°C for 5 minutes and photographed.
An important feature of using gold nanoparticles in colorimetric detection schemes is that the aggregation of AuNPs can be detected by UV/Vis spectroscopy. Since the absorption maximum of the 15 nm AuNPs used in this detection method is 520 nm, a decrease in absorbance at 520 nm can also be used to detect and quantitate aggregation. This was tested using reaction mixtures containing cell culture supernatants from DENV infected cells (Figure 7B). UV/Vis spectrophotometric analysis at A520 showed a decrease in absorbance when DDZ-M-AuNP positively detected DENV-2, suggesting the ability to quantitate these aggregation events. Mock or CHIKV infected cell culture fluids, or AuNPs tethered with the catalytically inactive DDZin-M do not elicit a detectible change in absorbance. These results show that our colorimetric DDZ-AuNP method for DENV detection possesses utility in a UV/Vis spectrophotometric platform.
DENV shares similar symptoms with other closely related mosquito-borne flaviviruses, such as Yellow Fever (YFV) [ 67 , 68 ], Japanese Encephalitis (JEV) [ 69 ], and Zika (ZV) [ 70 , 71 ] viruses. These viruses also co-circulate with DENV and are often misdiagnosed as dengue. Therefore, a DENV detection method must demonstrate the ability to distinguish DENV, from other mosquito-borne flaviviruses. Although the 5’-3’ CS domains are largely (but not fully) conserved among flaviviruses, the entire DDZ-M binding site is not conserved among all these flaviviruses as demonstrated by a sequence alignment of our DDZ-M binding site with corresponding regions in YFV, JEV, and ZV viruses. We also performed a experimental analysis of our DDZ-M-AuNP assay to verify its ability to distinguish DENV over other flaviviruses. Separate reaction mixtures were assembled as previously described (see Figure 4), except that artificial RNA substrates comprised of the 5’ 220 nucleotides of the YFV, JEV, ZV, and DENV genomes were used as targets (Figure 7C). This stretch of nucleotides included the highly conserved 5’ CS domain and the initial 74 bases of the capsid genes of each flavivirus. Aggregation of the DDZ-M-tethered AuNPs was evident only in the presence of the artificial DENV-2 RNA substrates and not YFV, JEV, or ZV. AuNPs tethered with the catalytically inactive DDZin-M did not aggregate in the presence of any flavivirus RNA substrate tested illustrating that mere binding of an RNA substrate is not enough to elicit an aggregation response by AuNPs. These results further validated the specificity of our DENV detection method.
Lastly, to be effective in epidemiological surveillance efforts, a DENV detection method must demonstrate the ability to detect each serotype independently of the other. An alignment of all four known DENV serotypes was performed to determine the ideal target sites for the design of serotype specific DNAzymes (Figure 7D) and appropriate targeting sequences were assembled (Table 1 ). Serotype- DNAzyme-tethered AuNPs were tested for their ability to detect viral genomic RNAs of DENV serotypes 1 through 4 (Figure 7E). AuNPs-tethered with either a serotype-specific DDZ or the multiple serotype detecting DDZ-M were placed in separate mixtures containing the DENV serotype indicated, 0.1% SDS to lyse virus particles, and 1.5 M NaCl (Figure 7E). Mixes were incubated at 37°C for 5 min.
Summary of active and negative control DDZ-AuNP devices
| Devices | Designed to detect |
Serotype detected
|
|||
|---|---|---|---|---|---|
| DENV-1 | DENV-2 | DENV-3 | DENV-4 | ||
| DDZ-M-AuNP
|
All Serotypes
|
+
|
+
|
+
|
+
|
| DDZ-1-AuNP
|
DENV-1
|
+
|
-
|
-
|
-
|
| DDZ-2-AuNP
|
DENV-2
|
-
|
+
|
-
|
-
|
| DDZ-3-AuNP
|
DENV-3
|
-
|
-
|
+
|
-
|
| DDZ-4-AuNP
|
DENV-4
|
-
|
-
|
-
|
+
|
|
Negative Control
|
|
||||
| DDZin-M-AuNP | None | - | - | - | - |
Left column lists the active (DDZ-M-AuNP and DDZ-1-AuNP through DDZ-4-AuNP) and inactive negative control (DDZin-M-AuNP) devices used in this report. The second column lists the serotype each device was designed to detect. Lastly, the right column lists the summarized results of the DENV detection devices and the negative control DDZin-M-AuNP. See Methods for description of DNAzyme design.
The DENV-1 serotype-specific DDZ-1-AuNP positively detected the DENV-1 serotype as signified by a distinctive red to clear/colorless color transition. As expected, DDZ-1-AuNP did not detect DENV-2, -3, or -4, illustrating the serotype-specific nature of this approach (Figure 7E). Likewise, each of the other serotype specific DNAzyme tethered AuNPs detected only the corresponding DENV serotype (Figure 7E and Table 1 ). These results demonstrated a DENV detection method that couples DNAzyme activity with AuNP aggregation to identify DENV in a serotype-specific manner. Cell culture supernatants containing the negative control CHIKV were added in lieu of DENV to further demonstrate the specificity of the serotype specific AuNPs and overall feasibility of our DENV detection assay. As expected, neither mock infected nor CHIKV infected cell culture supernatants yielded the red to clear color transition typically observed for the positive detection of DENV, showing our conjugated AuNPs were not influenced by cell or CHIKV derived oligonucleotides.
The sensitivity of our DENV detection system was assessed using standardized titers of DENV-2 (Figure 8). Titers of 10 1, 10 2,10 4 and 10 6 viruses/ml, as determined by TCID
Analysis of DDZ-AuNP sensitivity. A) DDZ-M-AuNP colorimetric assays were performed on DENV-2 NGC titers of 1 × 10 1, 1 × 10 2, 1 × 10 4, and 1 × 10 6 to determine the limits of detection. Samples were assembled as described in Methods, incubated at 37°C for 30 minutes and photographed. Results show that the DDZ-M-AuNP colorimetric assay is capable of detecting DENV-2 at a titer as low as 1 × 10 1. DDZ-M = anti-dengue virus DNAzyme, DDZin-M = inactive anti-dengue virus DNAzyme. B) Analysis of DDZ-AuNP sensitivity by UV/Vis spectrophotometry. Ten microliters (10 uL) of cell culture fluid from Mock, DENV-2 NGC (titers of 1 × 10 1, 1 × 10 2 1 × 10 6/mL), or CHIKV vaccine strain 181/25 (1 × 10 6/mL) infected C6/36 cells, or DENV serially diluted from 1 × 10 1 (Dil1 through Dil5) or were added to separate mixtures as described in A). UV/Vis spectrophotometric analysis was performed using the ND-1000 spectrophotometer at an absorbance of 520 nm. Absorbance measurements were graphed in log scale to illustrate sensitivity and accuracy of the colorimetric DENV detection method.
Positive DENV-2 detection was evident after only 5 minutes at 37°C, and demonstrated as little as 10 1 DENV/ml could cause a color transition, although the samples containing 10 1 and 10 2 did not transition to a very pale purple rather than completely clear. In addition, we calculated the amount of DENV RNA corresponds to approximately 0.6 μM (for 10 6/ml), 6 nm (for 10 4/ml), 0.6nM (for 10 2/ml), or 0.06 nM (for 10 1/ml) of DENV RNA per reaction.
Further assessment of the sensitivity of our colorimetric DENV detection assay was further assessed by UV/Vis spectrophotometry using standardized titers of DENV-2 (Figure 8B). Titers of 10 1, 10 2, and 10 6, viruses/ml, as determined by TCID
Simple and rapid diagnostic methods to screen mosquito and patient samples for the presence of viral pathogens can significantly facilitate diagnosis and treatment of virus borne diseases in field environments where sophisticated methods of virus detection are impractical. An ideal virus detection method must distinguish the target pathogen from other diseases exhibiting similar symptoms (such as malaria, leptospirosis, typhoid, typhus and Chikungunya), be highly sensitive during the acute stage of infection, provide rapid results, be inexpensive, easy to use, and stable at temperatures greater than 30°C for use in a field environment [ 61 ]. Furthermore, DENV detection methods must show utility in epidemiological surveillance and outbreak monitoring by allowing independent detection of each serotype, and must have the ability to distinguish between primary and secondary infection [ 61 ].
In light of the caveats and pitfalls of the virus detection methods currently in use [ 18 - 24 , 45 ], the aim of this research was to explore the utility of a multiple DENV serotype targeting DNAzyme, called DDZ-M, and DENV- serotype specific DNAzymes (Table 1 ), coupled to AuNPs for detecting DENV. DDZ was designed to target the most conserved region of the DENV genome that includes the 5’-3’CS (Figure 2A and 2B). DENV serotype-specific DNAzymes (designated DDZ-1 through DDZ-4) were engineered to bind regions of DENV that are conserved within each serotype. The demonstrated ability of DNAzymes to successfully target small stretches of RNA makes these catalytic oligonucleotides highly useful for targeting conserved regions of virus genomes. Our results suggest that DNAzyme targeting coupled with noncrosslinking AuNP aggregation satisfies many of the criteria required to have an ideal method for DENV detection.
While our DDZ-AuNP colorimetric detection system demonstrates the capacity to target the highly conserved DENV 5’ CS region, the utility of these molecules as detection agents requires a minimal subset of anti-DENV DNAzymes (DDZs) to be occupied for aggregation of AuNPs to occur. The high tolerance of DNAzymes to mismatched binding of the target oligonucleotides [ 74 , 75 ] makes DNAzymes ideal for detection of viruses because they will be able to detect many closely related variants. Prior studies have demonstrated aptazymes can detect synthetically produced segments of virus genomes [ 45 ]. We have demonstrated that under optimal reaction conditions the full length genome of DENV-2 can also be detected through the aggregation of DDZ-tethered AuNPs following the interaction of the DDZ component with the DENV-2 RNA genome.
Our anti-DENV DNAzyme (DDZ), when conjugated with AuNPs, readily detects its cognate target sequence within a synthetic 170 base segment of the DENV-2 NGC RNA corresponding to the 5’UTR, 5’CS and the 5’ 74 bases of the capsid open reading frame (Figure 4). Aggregation events result from deshielding AuNPs from sodium ions following DDZ binding to the synthesized DENV-2 target [ 76 ]. The DDZ-AuNP conjugate also detects purified viral RNAs or genomic RNA liberated from cell culture derived DENV-2 NGC virions. In our analyses we utilized cell culture supernatants instead of patient blood sample or infected mosquitoes because it is more convenient to determine optimal experimentation parameters (e.g. SDS and NaCl concentrations (Figure 5A and 5B, respectively) and limits of detection (Figure 8) using a less complex cell culture system. These results provide the first confirmation of effective DENV detection using our DDZ-AuNP assay, and represent for the first time a catalytic nucleotide-based method can be used to detect DENV in fluid. Subsequent analyses will be required to optimize procedures for applications with infected patient serum or mosquito tissues.
Previous studies using oligonucleotide-tethered AuNPs have determined optimal aggregation occurs with NaCl concentrations from 1.0 M to 1.5 M, while concentrations ≥ 2.0 M destabilized conjugated AuNPs [ 30 ]. In our hands, a NaCl concentration of 1.5 M allows full aggregation of DDZ-AuNP in the presence of 0.6 μM DENV-2 RNA (Figure 5A). We may infer that the color transition observed in samples containing DENV was not due to DNAzyme activity against the AuNP or non-specific interaction with cell derived oligonucleotides since the control solution containing 0 M NaCl did not yield a false positive result.
Furthermore, DDZ-AuNP aggregation in our DENV detection assays was not driven by the loss of AuNP stability in the presence of 10 mM MgCl
Sodium dodecyl sulfate (SDS) proved to be an effective, low cost, detergent for directly lysing virus particles in our assay [ 62 ]. SDS titration experiments on cell culture fluids containing DENV-2 NGC (Figure 5A). demonstrated a concentration of 0.5% (w/v) was sufficient to completely lyse DENV-2 particles without interfering with AuNP aggregation reactions. Addition of this detergent to the assay components has no effect on the cleavage or aggregation reactions.
Our DDZ-AuNP colorimetric assay is capable of distinguishing between DENV-2 NGC and CHIKV (Figure 7), two symptomatically related viral pathogens, and indicates the utility of this detection approach in regions of the world that are endemic to both DENV and CHIKV [ 63 - 65 ]. This increases the attractiveness and utility of the assay in epidemiological surveillance of dengue viruses in regions that are endemic to multiple pathogens that display similar symptoms. UV/Vis spectrophotometric analysis of these samples showed a fifty fold decrease in absorbance at 520 nm in the presence of DENV, demonstrating our DENV detection method has the sensitivity required for use with a spectrophotometer.
This DDZ-AuNP system allows for visual detection of DENV at titers as low as 10 1/mL, which translates to a concentration of 0.06nM DENV RNA (Figure 8A). This compares quite favorably to a previously reported RNA aptazyme-AuNP system that exhibits a sensitivity of 7.5nM [ 62 ]. Further assessment of the limits of DENV detection by UV/Vis spectrophotometric analysis (Figure 8B) demonstrates this assay displays sensitivity that is consistent with previous reports of RNA detection at sub-femtomole levels using gold nanoparticle detection systems [ 72 , 73 ]. Though detection of DENV RNAs at this low concentration is not physiologically relevant to what is present in posquitoes or humans, our ability to detect al this level demonstrates the power of AuNPs in detection schemes.
Moreover, despite the fact that we are detecting 1 × 10 6 TCID50 units, there are substantially more inactive virus particles present in a given sample [ 77 , 78 ]. Therefore, by adding SDS to lyse DENV particles the sensitivity of our DDZ-AuNP detection method is enhanced for real world applications. DENV infected patients exhibit titers of 10 7 to 10 8.5 TCID
We have to date demonstrated that our multi-DENV serotype detecting DDZ-M-AuNP device can detect all four DENV serotypes directly from cell culture fluid without sample processing (Figure 7E). Furthermore, serotype-specific DDZ-tethered AuNPs have demonstrated utility in detecting each of the corresponding four DENV serotypes in a serotype specific manner (Figure 7E). For example, DDZ-1 tethered AuNPs detected the presence of DENV-1, and only the DENV-1 serotype, due to the designed specificity of the DDZ-1 DNAzyme to a region in the DENV-1 Capsid gene that is fully conserved solely among the DENV-1 serotype. The other serotype specific DDZ-tethered AuNPs possess this same feature in the detection of their corresponding DENV serotype (Figure 7E, see results summarized in Table 1 ). Full development of this system will provide a valuable method for the detection of DENV in a serotype specific manner in mosquito populations leading to enhanced speed and accuracy of epidemiological surveillance.
The DDZ-AuNP assay’s simplicity provides distinct advantages over other virus detection methods. The assay can be packaged as a pre-mixed reaction solution in eppendorf tubes, and may be performed without any specialized equipment or training. Furthermore, this assay is inexpensive, costing about $0.80 per sample, as compared to serological testing or PCR-based methods which can cost $2 per sample or more to perform. Assay components are stable for months at room temperature [ 83 ], and exhibit stability at temperatures greater than 30°C.
We anticipate further development of this assay will enable sensitive detection and discrimination of individual DENV serotypes in mosquito populations and patient derived samples as well as other virus derived RNAs. Detection prior to the onset of symptoms could allow more effective diagnosis and treatment of infected patients, and more rapid recovery from the disease. The simplicity of the assay makes it ideal as a means of early surveillance to target locations for more effective mosquito suppression strategies.
The results presented here show that the DDZ-M-AuNP, designed to be active against all forms of dengue virus, is capable of effectively detecting the DENV 2-NGC genome in a sequence specific manner. Serotype specific DNAzymes tethered to AuNPs demonstrate utility in the independent identification of DENV serotypes. Coupling DNAzyme catalysis with gold nanoparticle aggregation provides an attractive alternative to other DENV detection approaches for the identification of DENV in transformed mosquito cells and tissues.
Thiol-modified and unmodified DNAzymes were synthesized and desalted by Life Science Technologies (Grand Island, NY, USA). The oligoribonucleotide target was synthesized and HPLC-purified by Life Science Technologies. Quantification of these oligonucleotides was performed with the ND-1000 spectrophotometer from NanoDrop (Wilmington, DE). Gold colloidal solutions containing 1.6 × 10 12 particles/mL gold nanoparticles (AuNPs) with a diameter of 15 nm were purchased from Cytodiagnostics (Burlington, ON, CA).
Ae. albopictus C6/36 cells were obtained from ATCC, and maintained in Leibovitz’s L-15 media (Atlanta Biologicals) supplemented with 10% FBS (Atlanta Biologicals), 10% TPB (triptose phosphate broth; Invitrogen/Gibco), penicillin G (100 U/ml; Invitrogen/Gibco) and streptomycin (100 U/ml; Invitrogen/Gibco). The C6/36 cells used in this study were maintained in a 28°C incubator and passaged every 4 days. Viral stocks were prepared as previously described [ 78 ].
The DENV strains and Genbank GenInfo identifiers for the four serotypes used in this study are as follows: DENV type 1 Hawaii: DQ672564.1, DENV type 2 strain New Guinea C (NGC): AF038403.1, DENV type 3 strain ThD3 0010 87(strain H87): AY676352.1, DENV 4 strain DENV-4/SG/06K2270DK1/2005 (strain H241): GQ398256.1.
DENV sequence data was obtained from the national center of biotechnology information (NCBI). Sequences representative of all four serotypes of dengue were aligned using ClustalX [ 54 , 55 ]. The aligned sequences comprise the following genbank GenInfo identifiers: 12018173, 12018169, 12018171, 12659201, 2909798, 2909788, 2909786, 2909796, 6841603, 6841595, 6841605, 6841591, 6841601, 6841597, 6841593, 6841599, 6841587, 6841585, 6841589, 1000740, 1000738, 2909784, 1000736, 4926937, 4926935, 4926927, 4926929, 4926931, 2909794, 2909792, 1000742, 4926933, 2155257, 2723944, 323447, 6581076, 6581078, 2723942, 323449, 323650, 18644123, 1864412, 11119731, 19744844, 18644125, 18644127, 18643733, 4337012, 13386495, 1881708, 19071809, 13926152, 9280544, 14585842, 4926947, 4926939, 323654, 4926945, 4926943, 7329983, 7329981, 13540386, 14328931, 14485523, 323660, 17129645, 22901065, 22901063, 22901061, 1854040, 1854038, 1854036, 17129647, 24417519, 24417517, 24417515, 27656962, 24417513, 19071807, 14195698, 8927332, 14328929, 12711599, 323468, 25992053, 25992047, 25992041, 25992029, 25992025, 25992055, 25992033, 19071811, 25992043, 25992039, 25992037, 25992051, 25992031, and 25992057.
The 5’ arms of DDZ-M and DDZin-M (Table 2 ) were designed to bind to nucleotides 150 to 158 of the DENV genome. The 3’ arms were designed to bind to the 5’ end of the target region of the DENV genome that corresponds to nucleotides 140 to 148. These 5’ and 3’ arms of facilitated DDZ cleavage of the substrate DENV RNA between the purine-pyrimidine dinucleotide motifs G149 and C150.
Nucleotide sequences of active and negative control DNAzymes and corresponding targets
| DNAzyme | 5’Arm (5’-- > 3’) | 3’Arm (5’-- > 3’) | Catalytic core | Target |
|---|---|---|---|---|
| DDZ-M
|
TTTCTCTCG
|
GTTTCAGCA
|
GGCTAGCTACAACGA
|
TGCTGAAACGCGAGAGAAA
|
| DDZ-1
|
ATCGCTCCA
|
TCTTCTTGA
|
GGCTAGCTACAACGA
|
TCAAGAAGAATGGAGCGAT
|
| DDZ-2
|
AAAGGCGTA
|
TTCTCGCCT
|
GGCTAGCTACAACGA
|
AGGCGAGAAATACGCCTTT
|
| DDZ-3
|
TAGCCAAGA
|
TCCTGCTGT
|
GGCTAGCTACAACGA
|
ACAGCAGGAGTCTTGGCTA
|
| DDZ-4
|
GTTGGTTCA
|
TTTTCCAGA
|
GGCTAGCTACAACGA
|
TCTGGAAAAATGAACCAAC
|
|
Negative Control
|
||||
| DDZin-M | TTTCTCTCG | GTTTCAGCA | AGCAACATCGATCGG | TGCTGAAACGCGAGAAA |
Left column lists the active (DDZ-M) and inactive (DDZin-M) DNAzymes used in this report. The second and thrid columns list the sequences of the 5’and 3’ binding arms of the catalytically active DNAzymes and the inactive DDZin-M, respectively. Also shown are the sequences of the catalytic cores of each DNAzyme. The right column lists the nucleotide sequence each binding arm binds to where applicable. All sequences are displayed in a 5’ to 3’ direction. See Methods for description of DNAzyme design.
The DDZ target site was selected by scanning the 5’CS domain for one of the purine-pyrimidine dinucleotide motifs required for DNAZyme catalysis [ 36 ]. The primary criterion for selection was that a purine-pyrimidine motif located within the target site must be present in all strains of a given DENV serotype. Another important criterion for selecting suitable sites for DDZ cleavage was that the length of conserved flanking arms be long enough to insure specificity of the DNAzyme for the target site. The 5’ and 3’ arms of each DDZ were 9 bases in length since this was determined to be optimal for DNAzyme catalysis and provides a sufficient level of specificity to insure minimal off-target effects [ 36 ].
Preparation of DDZ-M-AuNP was performed as previously described [ 83 ] with a few modifications. The DTT-reduced thiol-DDZ-M 5’-SH-(CH2)6-d(TTTCTCTCGGGCTAGCTACAACGAGTTTCAGCA)-3’ (SH-DDZ-M) was purified by ethanol precipitation. 3 ml of AuNP and 5 mM acetate buffer (pH 5.2) were transferred to a glass scintillation vial, capped and incubated for 24 hours at room temperature. Following incubation 5 mM Tris acetate (pH 8.2) buffer and 100 mM NaCl were added and the resulting mixture was incubated at room temperature for an additional 24 hours. These functionalized particles (500 μl) were transferred into 1.7-ml microcentrifuge tubes and centrifuged at 16,110 g at room temperature for 15 min to remove unreacted SH-DDZ-M. The nanoparticles were redispersed in 1 mL of buffer containing 100 mM NaCl, 25 mM Tris acetate, (pH 8.2) and 0.01% SDS, centrifuged again at 16,110 g at room temperature for 15 min. The supernatant was removed and the nanoparticles were dispersed in 500 μl of buffer containing 300 mM NaCl and 25 mM Tris acetate (pH 8.2), and re-centrifuged for 15 min to remove the remaining unreacted SH-DDZ-M. The cleaned DDZ-M-AuNP were redispersed into 200 μL of buffer containing 100 mM NaCl, 25 mM Tris acetate, (pH 8.2) and 0.05% SDS. This same procedure was followed for the coupling of DENV serotype-specific DTT-reduced DNAzymes: thiol-DDZ-1 5’-SH-(CH2)6-d(AGCCAxAAAGGCTAGxCTACAACGATCCTGCTG)-3’(SH-DDZ-1), thiol-DDZ-2 5’-SH-(CH2)6- d(AAGGCGTAGGCTAGCTACAACGATTCTCGCC)-3’ (SH-DDZ-2), thiol-DDZ-3 5’-SH-(CH2)6-d(AGCCAAGAGGCTAGCTACAACGATCCTGCTG)-3’ (SH-DDZ-3), and thiol DDZ-4 5’-SH-(CH2)6-d(TTGGTTCGGCTAGCTACAACGATTTTCCAG)-3’ (SH-DDZ-4).
DDZ-AuNPs (1 × 10 5/mL) were combined in a 1.5 mL eppendorff tube with 10 mM MgCl
This analysis was performed as previously described [ 30 ]. A mixture composed of 1 μL of DDZ-tethered AuNPs, 50 mM Tris–HCl (pH 7.5), and increasing concentrations of MgCl
DENV RNA was isolated from DENV infected Ae. albopictus C6/36 cells using the QiaAmp viral RNA Mini Kit (Qiagen) according to the manufacturer’s protocol. 10 μM of eluted DENV RNA was incubated with 1 × 10 5 DDZ-AuNP/ml for 30 min at 37°C. 15 ul of this reaction mixture was added to a RT-PCR mix (Life Science Technologies) containing heterologous and random hexametric primers to amplify the digested fragments. These RT-PCR fragments were then separated on 1.75% agarose gels.
Ten microliters (10 μl) of cell suspension containing 1 × 10 6 DENV-2 NGC/mL, as determined by TCID
DENV -2 NGC RNA were isolated from Aedes albopictus C6/36 cells using the Qiamp Viral RNA mini kit, and added at a concentration of 0.6 μM (~10μL) to a reaction mixture containing 150 mM Tris–HCl (pH 7.5), 10 mM 10 mM MgCl
Ten microliters (10 uL) of cell culture fluid containing 1 × 10 6/mL DENV-2 NGC or, as a negative control, CHIKV vaccine strain 181/25 [ 84 ] was added to a mixture containing 150 mM Tris–HCl (pH 7.5), 10 mM 10 mM MgCl
DENV-2 NGC of the titers indicated (Figure 8) were produced as follows. A titer of 1 × 10 6/mL was obtained following inoculation of Ae. albopictus C6/36 cells with 0.1 MOI and incubated at 28°C for 6dpi. DENV-2 NGC were grown to titers of 1 × 10 4/mL and 1 × 10 2/mL at 3dpi and 6dpi, respectively, following inoculation of Vero cells with MOI of 0.1. DENV-2 NGC at a titer of 1 × 10 1/mL were produced by serial dilution of the 1 × 10 2/mL stock. Titers were determined by TCID
The DENV-2 NGC titers described above served as substrates for DDZ-AuNP colorimetric assays to determine their limits of DENV detection. Ten microliters (10 μl) of each dilution stock was added to a buffered reaction mix containing 150 mM Tris–HCl (pH 7.5), 10 mM 10 mM MgCl
The coupled DNAzyme-gold nanoparticle detection technology is part of a pending patent application, numbers USSN 16/835,173 and USSN 61/835,758.
JRC engineered the DENV specific DNAzyme tethered AuNP approach and performed all RT-PCR and detection assays. VB performed all TCID50 analysis of DENV and CHIKV virus stocks to determine titer and confirm identity. CAK maintained all virus stocks, and TSF maintained all cell cultures used in this research. MJF provided technical assistance. The manuscript was prepared by JRC and MJF. All authors read and approved the final manuscript.
We would like to thank Dr. Hsueh-Chia Chang (University of Notre Dame) and his lab for their suggestions and expert advice. This study was supported by the NIH/NIAID RO1AI048561 to MJF.


 More...
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| |
| BLACKBONE_API bool | Suspended () |
| | Check if thread is suspended More...
|
| |
| BLACKBONE_API bool | GetContext (_CONTEXT32 &ctx, DWORD flags=CONTEXT_ALL, bool dontSuspend=false) |
| | Get WOW64 thread context More...
|
| |
| BLACKBONE_API bool | GetContext (_CONTEXT64 &ctx, DWORD flags=CONTEXT64_ALL, bool dontSuspend=false) |
| | Get native thread context More...
|
| |
| BLACKBONE_API bool | SetContext (_CONTEXT32 &ctx, bool dontSuspend=false) |
| | Set WOW64 thread context More...
|
| |
| BLACKBONE_API bool | SetContext (_CONTEXT64 &ctx, bool dontSuspend=false) |
| | Set native thread context More...
|
| |
| BLACKBONE_API bool | Terminate (DWORD code=0) |
| | Terminate thread More...
|
| |
| BLACKBONE_API bool | Join (int timeout=INFINITE) |
| | Join thread More...
|
| |
| BLACKBONE_API DWORD | ExitCode () const |
| | Get thread exit code More...
|
| |
| BLACKBONE_API int | AddHWBP (ptr_t addr, HWBPType type, HWBPLength length) |
| | Add hardware breakpoint to thread More...
|
| |
| BLACKBONE_API bool | RemoveHWBP (int idx) |
| | Remove existing hardware breakpoint More...
|
| |
| BLACKBONE_API bool | RemoveHWBP (ptr_t ptr) |
| | Remove existing hardware breakpoint More...
|
| |
| BLACKBONE_API void | Close () |
| | Close handle More...
|
| |
|
BLACKBONE_API bool | operator== (const Thread &other) |
| |
|
BLACKBONE_API Thread & | operator= (const Thread &other) |
| |
| int blackbone::Thread::AddHWBP |
( |
ptr_t |
addr, |
|
|
HWBPType |
type, |
|
|
HWBPLength |
length |
|
) |
| |
Add hardware breakpoint to thread
- Parameters
-
| addr | Breakpoint address |
| type | Breakpoint type(read/write/execute) |
| length | Number of bytes to include into breakpoint |
- Returns
- Index of used breakpoint; -1 if failed
| void blackbone::Thread::Close |
( |
| ) |
|
| uint64_t blackbone::Thread::execTime |
( |
| ) |
|
Get total execution time(user mode and kernel mode)
- Returns
- Total execution time
| DWORD blackbone::Thread::ExitCode |
( |
| ) |
const |
Get thread exit code
- Returns
- Thread exit code
| bool blackbone::Thread::GetContext |
( |
_CONTEXT32 & |
ctx, |
|
|
DWORD |
flags = CONTEXT_ALL , |
|
|
bool |
dontSuspend = false |
|
) |
| |
Get WOW64 thread context
- Parameters
-
| ctx | Returned context |
| flags | Context flags. |
| dontSuspend | true if thread shouldn't be suspended before retrieving context |
- Returns
- true on success
| bool blackbone::Thread::GetContext |
( |
_CONTEXT64 & |
ctx, |
|
|
DWORD |
flags = CONTEXT64_ALL , |
|
|
bool |
dontSuspend = false |
|
) |
| |
Get native thread context
- Parameters
-
| ctx | Returned context |
| flags | Context flags. |
| dontSuspend | true if thread shouldn't be suspended before retrieving context |
- Returns
- true on success
| BLACKBONE_API HANDLE blackbone::Thread::handle |
( |
| ) |
const |
|
inline |
Get thread handle
- Returns
- Thread hande
| BLACKBONE_API DWORD blackbone::Thread::id |
( |
| ) |
const |
|
inline |
Get thread ID
- Returns
- Thread ID
| bool blackbone::Thread::Join |
( |
int |
timeout = INFINITE | ) |
|
Join thread
- Parameters
-
- Returns
- true on success
| bool blackbone::Thread::RemoveHWBP |
( |
int |
idx | ) |
|
Remove existing hardware breakpoint
- Parameters
-
- Returns
- true on success
| bool blackbone::Thread::RemoveHWBP |
( |
ptr_t |
ptr | ) |
|
Remove existing hardware breakpoint
- Parameters
-
- Returns
- true on success
| bool blackbone::Thread::Resume |
( |
| ) |
|
Resumes thread.
- Returns
- true on success
| bool blackbone::Thread::SetContext |
( |
_CONTEXT32 & |
ctx, |
|
|
bool |
dontSuspend = false |
|
) |
| |
Set WOW64 thread context
- Parameters
-
| ctx | Context to set |
| dontSuspend | true if thread shouldn't be suspended before retrieving context |
- Returns
- true on success
| bool blackbone::Thread::SetContext |
( |
_CONTEXT64 & |
ctx, |
|
|
bool |
dontSuspend = false |
|
) |
| |
Set native thread context
- Parameters
-
| ctx | Context to set |
| dontSuspend | true if thread shouldn't be suspended before retrieving context |
- Returns
- true on success
| uint64_t blackbone::Thread::startTime |
( |
| ) |
|
Get thread creation time
- Returns
- Thread creation time
| bool blackbone::Thread::Suspend |
( |
| ) |
|
Suspend thread
- Returns
- true on success
| bool blackbone::Thread::Suspended |
( |
| ) |
|
Check if thread is suspended
- Returns
- true if suspended
| blackbone::ptr_t blackbone::Thread::teb |
( |
_TEB32 * |
pteb = nullptr | ) |
const |
Get WOW64 TEB
- Parameters
-
- Returns
- TEB pointer
| blackbone::ptr_t blackbone::Thread::teb |
( |
_TEB64 * |
pteb = nullptr | ) |
const |
Get Native TEB
- Parameters
-
- Returns
- TEB pointer
| BLACKBONE_API ptr_t blackbone::Thread::teb |
( |
| ) |
const |
|
inline |
Get TEB
- Returns
- TEB pointer
| bool blackbone::Thread::Terminate |
( |
DWORD |
code = 0 | ) |
|
Terminate thread
- Parameters
-
- Returns
- true on success
| BLACKBONE_API bool blackbone::Thread::valid |
( |
| ) |
const |
|
inline |
Check if thread exists
- Returns
- true if thread exists
The documentation for this class was generated from the following files:
- C:/Users/Ton/Documents/visual studio 2013/projects/blackbone/src/BlackBone/Process/Threads/Thread.h
- C:/Users/Ton/Documents/visual studio 2013/projects/blackbone/src/BlackBone/Process/Threads/Thread.cpp


3 references to ReceiveCommandOutputEx
System.Management.Automation (3)
engine\remoting\client\remotingprotocolimplementation.cs (1)
511
case TransportMethodEnum.
ReceiveCommandOutputEx
:
engine\remoting\fanin\WSManTransportManager.cs (2)
3724
new TransportErrorOccuredEventArgs(e, TransportMethodEnum.
ReceiveCommandOutputEx
);
3751
TransportMethodEnum.
ReceiveCommandOutputEx
,


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--- layout: documentation title: ActionAliasesType ---
The ActionAliasesType enables identification of other potentially used names for this Action.
| Field Name | Type | Description |
|---|---|---|
| Action_Alias1..n | string |
The Action_Alias field is optional and enables identification of a single other potentially used name for this Action. |


a https://en.wikipedia.org/wiki/ICloud_leaks_of_celebrity_photos
b https://en.wikipedia.org/wiki/PRISM_(surveillance_program)
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At the Dry Tortugas During the War
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THE
BIG INDIAN
The following is
excerpted from an undated letter written by Doctor N. E. Henthorn (who died
around 1880) to John B. Noll, Esquire.
"In 1831, I was returned home from Cincinnati by land. I
stopped overnight in the town of Reading, 12 miles from the city and stayed at
Jackson s Tavern. When the landlord asked where I was from, He said his father
and an old Indian would like to talk to me. I went to their room and Mr.
Jackson, Sr., said he knew my grandfather at the old blockhouse in Wheeling. He
further stated that, at the time Boggs was killed at Boggs Island, the Indians
were pursued by the white men.  He said that the Indian had lived with him
ever since and that he was with him in the War of 1812 under General Harrison.
The Indian told me that the Indian name for Sunfish Creek was Buckchitawa and
the Indian name for Opossum Creek was Eagle Creek. He further told me that a
big Indian was killed at Buckchitawa at about the same time Marietta was
settled in 1788. The Indians had a white prisoner whom they forced to decoy their
boats to the shore. A small boat was descending the river carrying white people
when the prisoner was placed under the bank to tell those in the boat that he
had escaped captivity and to come to the shore and take him in. The Indians
were concealed but the big Indian stuck his head out from behind a large tree
and, as a result, it was pierced by a bullet from the gun of the steersman of
the boat. The Indians cried out, "Wetzel, Wetzel!" and fled. The
prisoner was never seen again. The Indians returned the next day and buried the
big Indian.
"
When Chester Bishop was digging a cellar for Asahel Booth in Clarington many
years ago, he came across a skeleton. The bones were carefully removed by Dr.
Richard Kirkpatrick. From his measurements, the height of the man would have
been about eight feet and five inches when he was alive. The Indian at Mr.
Jackson s further told me that there was lead on Opossum Creek, on Sunfish
Creek, and on Captina Creek but that the veins were thin. "
Taken from History of Monroe County, Ohio by the Woodsfield
Chamber of Commerce, 1963.
Sunfish Creek and Oppossum Creek are located in present day Salem
Township, Monroe County, Ohio. Both empty into the Ohio River, where this
confrontation occurred.
The Wetzel reference undoubtedly refers to the famed Lewis Wetzel, who
was called Deathwind by the Indians. Whether he or one of his brothers were in
the boat or if they assumed the accurate shot and rapid recognition of the
ambush suggested it was a Wetzel cannot be determined.
Click here for the Short-Cut Table of Contents


Derek Eder
@derekeder
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Derek Eder
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--- layout: post type: post status: publish tags: - bash - condition - fedora - shell - Technical - terminal - update - yum meta: _wpbitly: http://q.bibhas.in/H70UOA _edit_last: "1" published: true title: Selective/Conditional Update with Yum --- The first thing I noticed after installing Fedora 14 was that there are updates available for my packages. And not a few, more than 400 updates! Sizing about 500mb!  Updating all the packages in one go will make me keep this system on for about 5 hours. Thats not possible for me. So I needed a way to update few packages everytime and keep doing that whenever I find time.Now I can open the update manager, untick about 350 package the first time to update first 50 package and go on like that.. Sounds ridiculous, right? So ticking-unticking aint an option, lets look for something new. Terminal is always there, lets use it. I thought of using conditions with the yum command. Like defining a number, so that first that number of packages will be updated. But that'd most probably require me to write a complicated shell script which I'm not intending to do right now. Next option, lets try to find if any condition can be applied to packages' name and update only the matching ones. This idea seem to have an easy way to deploy. I found that you can use wildcard (*) to the yum update command. Now that's cool! it lets me to select packages based on a portion of their name. Like, if I want to update only the packages that start with a, all I need to do is use this command -
# yum update a*Or if I want to update all the packages that has the phrase py in them, I'd use the command -
# yum update *py*I can also define more than one condition with a space as delimiter, like -
# yum update 'a*' '*bas*'That's it! I got my way to update few packages at a time. :D
Note: Before using Yum, install the yum plugin yum-fastestmirror. Saves time finding the the mirrors that'll transfer data faster. :)


{% extends "layout-chs.html" %} {% block page_title %} Accounting policies {% endblock %} {% block proposition_header %} {% endblock %} {% block header_class %} {% endblock %} {% block content %} {% include "includes/phase_banner_beta.html" %} Back {% include 'includes/error.html' %} 3 Balance sheet notes Accounting policies This page describes the methods and measurements you've used to prepare your company accounts. Choose the statement that reflects the way your accounts were prepared: What's this?Choose the statement that reflects the way your accounts were prepared This policy is about the accounting standards you've prepared your accounts to. As a small company you're likely to have prepared your full accounts for shareholders or members using the simpler provisions of Section 1A (Small Entities) of Financial Reporting Standard 102. Your accounts should include a statement about the basis of measurement and preparation. Basis of preparation These financial statements have been prepared in accordance with the provisions of Section 1A (Small Entities) of Financial Reporting Standard 102 These accounts have been prepared in accordance with another standard Enter the basis of measurement and preparation for your accounts: Do your prepared accounts include any additional accounting policies? For example, "Turnover policy" or "Tangible fixed assets depreciation policy" Do your prepared accounts include any additional accounting policies? Yes No Complete all items that are included in your prepared accounts Turnover policy What's this?Turnover policy This policy describes how the turnover figure in your profit and loss account was calculated. For example, 'The turnover represents amounts receivable for goods and services net of VAT and trade discounts'. Turnover policy Tangible fixed assets depreciation policy What's this?Tangible assets depreciation policy If you have tangible (physical) fixed assets, this policy describes how the depreciation (reduction in value) of each asset is calculated over its expected useful lifetime. For example, 'Annual depreciation of computer equipment is calculated at 25% of cost to write off the cost of the asset, less its residual value, over its estimated useful economic lifetime'. Tangible assets depreciation policy Intangible fixed assets amortisation policy What's this?Intangible assets amortisation policy If you have intangible (non-physical) fixed assets, this policy describes how the amortisation (reduction in value) of each asset is calculated over its expected useful lifetime. Amortisation is similar to the depreciation value of a tangible (physical) asset. For example, 'Annual amortisation of goodwill is calculated at 25% to write off the cost of the asset, minus its residual value, over its estimated useful lifetime'. Intangible assets amortisation policy Valuation information policy What's this?Valuation information policy If you have stocks or work in progress, this policy describes how they are valued. Stocks should be valued at the lower of either cost or ‘net realisable value’ (the realistic resale value minus the cost of selling it). For example, 'Stocks and work in progress are valued at the lower of cost and net realisable value, after making allowances for obsolete and slow-moving items. Cost includes all direct expenditure and overheads'. Valuation information policy Other accounting policies What's this?Other policy Use this policy to report other policies you may have that aren’t covered by the policies above, such as: depreciation of assets obtained under hire purchases or leases deferred taxation pensions or retirement benefits Other policy {% endblock %}
This page describes the methods and measurements you've used to prepare your company accounts.
What's this?Choose the statement that reflects the way your accounts were prepared This policy is about the accounting standards you've prepared your accounts to. As a small company you're likely to have prepared your full accounts for shareholders or members using the simpler provisions of Section 1A (Small Entities) of Financial Reporting Standard 102. Your accounts should include a statement about the basis of measurement and preparation.
This policy is about the accounting standards you've prepared your accounts to. As a small company you're likely to have prepared your full accounts for shareholders or members using the simpler provisions of Section 1A (Small Entities) of Financial Reporting Standard 102. Your accounts should include a statement about the basis of measurement and preparation.
Complete all items that are included in your prepared accounts
This policy describes how the turnover figure in your profit and loss account was calculated.
For example, 'The turnover represents amounts receivable for goods and services net of VAT and trade discounts'.
If you have tangible (physical) fixed assets, this policy describes how the depreciation (reduction in value) of each asset is calculated over its expected useful lifetime.
For example, 'Annual depreciation of computer equipment is calculated at 25% of cost to write off the cost of the asset, less its residual value, over its estimated useful economic lifetime'.
If you have intangible (non-physical) fixed assets, this policy describes how the amortisation (reduction in value) of each asset is calculated over its expected useful lifetime. Amortisation is similar to the depreciation value of a tangible (physical) asset.
For example, 'Annual amortisation of goodwill is calculated at 25% to write off the cost of the asset, minus its residual value, over its estimated useful lifetime'.
If you have stocks or work in progress, this policy describes how they are valued. Stocks should be valued at the lower of either cost or ‘net realisable value’ (the realistic resale value minus the cost of selling it).
For example, 'Stocks and work in progress are valued at the lower of cost and net realisable value, after making allowances for obsolete and slow-moving items. Cost includes all direct expenditure and overheads'.
Use this policy to report other policies you may have that aren’t covered by the policies above, such as:


Chester Cheetah
Home
History
Commericals
Fan Art
Chester commericals
These old Chester Cheetah commericals are a perfect example of how cool he is


Eric Versteeg
Student since February 28, 2002
After I retired a few years ago, I decided I wanted to learn to play the piano. I had never played the piano, but it was my good fortune to have Tony Onorati as my teacher from the beginning of my musical sojourn.
Tony has been a wonderful teacher. He is knowledgeable, patient, and good humoured. Tony has an excellent grasp of what my objectives have been, and I am still taking piano lessons. He has structured his lessons to challenge me and also to provide me with exposure to a broad variety of piano music.
Given the very positive experience I have had with my piano lessons, I have no hesitation in recommending Tony as a piano teacher.
Gaetano Geretto
Student since January 19, 2006
I have learned a lot from Tony Onorati in the many years I have worked with him. He is a diligent teacher, who gets the most from his students by challenging them and making the work fun!
He encourages his students and offers appropriate praise. He ensures his students work hard on technique and helps students apply that technique to their pieces. He prepares his students very well for the RCM examinations.
I feel privileged and honoured to have Tony as my teacher and I would highly recommend him as a great piano teacher.
Samantha Lim
Student since July 6, 2006
I have been taking piano lessons with Tony for nine years, and he has taught me a lot. I have completed various repertoire and theory examinations with success. In addition to learning the necessary Royal Conservatory of Music songs, Tony also gives me popular songs for enjoyment. His patience and encouragement has motivated me to continue with piano.
Tony Onorati is by far the best piano teacher I've had. I am excited to learn more under his guidance for years to come.
Kelly Tan
Student since March 4, 2007
I have been getting piano lessons from Tony since 2007, of which then was presented to me as a supposed birthday gift. As a young child back then, I did not expect to take full pleasure in this. But now, 8 years later, I have come to appreciate it and I am able to learn about music and play the piano well.
Tony is a great piano teacher! Not only are the lessons informative but they are also enjoyable. It is the gateway to enjoying music.
Lorraine Robinson
Student since October 24, 2006
When I started piano lesson in 2006, all I knew about piano is that I wanted to learn to play. Within a year, I was playing a varied selection of music arranged by Tony and music I enjoyed playing.
Tony has infinite patience, is engaging and lessons are never boring and has a rapport with students of all ages. All he asks of his students is they practice with a capital P.
Janet Neilson
Student since September 19, 2012
Tony is a fabulous teacher. He has all the qualities one needs (and of course benefits from) in a teacher -- years of experience, unbelievable patience, exactness, a vast musical knowledge base, and talent himself.
He is able to convey clearly where the challenges in a piece might emerge and the rationale for learning a particular scale, piece of music or musical style. His weekly instructions are meticulously laid out; musical pieces are clearly marked for fingering and timing, and homework is negotiated with respect for the external demands of life.
As a beginner adult student who felt "all thumbs and very dumb" when I began this journey, I am surprised at the progress I have made. I now play pieces that I never imagined I possibly could, and I actually sometimes enjoy listening to myself -- certainly the dog no longer covers his ears.
Tony is an extremely nice person, and it is a pleasure to embark on this musical journey with him as my guide.
Simone Cattana
Student since July 12, 2011
I have been studying with Tony for 4 years. I just completed my RCM Grade One Exam. I got First Class Honors with Distinction. I know I could not have achieved this outstanding grade without Tony's help.
The week of my exam I was at home sick. Tony scheduled a time to call me to review everything. It really helped.
I also love that he has a concert at the end of the year. I get to play a song I love, and sometimes get to sing too! (I love being on stage). I also learn Christmas songs that I can play over the holidays, and the entire family joins in to sing along. For these reasons I don't feel I am always working hard just to pass the RCM examinations or learning new theory.
Angela Krstic
Student since September 26, 2013
I have learned a lot about piano since I have been taking lessons with Tony. I really like how he asks me how I am before every class. He is very friendly and polite.
I also like how everything is well kept and organized. He is very patient while I am learning a new song and I am making errors. Overall, I am having a great experience taking lessons with Tony, and I am glad I came to learn music at Da Capo studio.
Aline Huynh
Student since July 6, 2009
As a student studying both practical and theoretical piano under Tony Onorati for the past six years, I have been privileged with his patient teaching style and encouragement. His ability to understand his students and accommodate to each of our different learning styles is exemplary.
He is patient and kind to all his students, allowing us to trust him and thereby, enhancing our learning experience and maximizing our success. Tony has made my goals and success his primary concern as he encourages me to strive for greatness. Not only is Tony a kind-hearted and admirable teacher who has given me the necessary skills to succeed musically, he has also taught me the importance of hard work and motivation; these are the skills that are not only necessary in piano, but necessary to accomplish great things throughout my life.
Katarina Pupovac
Student since October 4, 2007
I've been at Da Capo for 8 years and I've learned so much from my time here. Not only is Da Capo a great place for musicians of any skill level, it also incorporates aspects of music that each individual student enjoys to make the experience more fun.
The memories I have made and the skills I've acquired during my time here will last me a lifetime and I look forward to my sessions here each week.
Luca Mancini
Student since October 8, 2009
I have been taking piano classes with Tony for 6 years. The thing I like most about my piano lessons are the year-end concert. I love performing for people.
Apart from learning all my Royal Conservatory course, I also learn to play pop songs. Soon I will be taking my first RCM Exam. I feel Tony has prepared me well and I am confident I will do very well. Occasionally, Tony gives me surprise in-class theory tests, and while I do my test, Tony gives me a little treat.
I really enjoy taking music lessons with Tony because he makes learning fun. He always has a smile on his face.
Ken Viljoen
Student since October 3, 2013
Two years ago, at the age of 75, I started to learn how to play the piano. I have found that Tony is patient, perceptive, knowledgeable, and very encouraging.
He follows progress step by step, and gives an honest opinion of my progress. Be aware that to take lessons with Tony, you must practice!


--- layout: post title: Getting Michael Sandler's Barefoot Running Book in the UK date: 2010-05-19 17:31:53.000000000 +01:00 tags: - running - barefoot - book type: post published: true ---
I've just attempted to get my hands on Michael Sandler's book "Barefoot Running: How to Run Light and Free by Getting in Touch with the Earth" and it's proving to be a bit of a mission, unless my money tree starts growing, and quickly.
The book has just been released and the reviews of the book are starting to popup all over the barefoot and minimalist running websites. I consider myself to be a seasoned barefoot runner now, but I'm eager to have a read through this book to see how closely my experience has been to that of the author's and also to see if I've missed some "magic trick" or other little useful snippet that would help improve my barefoot running.
So, the first place I hit was Amazon.com. I've got a gift voucher that isn't transferable to Amazon.co.uk so thought I'd use it for this book. Nope. Sadly, the book isn't sold directly by Amazon, but rather by the publisher and they won't ship to the UK via Amazon. The book also isn't available via Amazon.co.uk, nor any of the other European equivalents. So it's to the book's site itself.
Well, that's a bit of a dead end too - the cost of the P&P is nearly as much as the book. The book costs $24.95 and P&P to the UK is $19.95. Add to this the fact the Pound is sliding against the Dollar, by the time the book arrives in the UK I could soon find myself in the position where the book costs more than £18 and thus becomes liable to import duties (not VAT as books are exempt) and thus I'd have paid more than double the price for the book.
Eager to find out if there is anyway I can get this in the UK without it costing me twice as much, I've used the book site's contact form to find out. I'll update you when I hear back.
In the mean time, if anyone knows of somewhere in Europe that is selling this book, then please let me know.
Update - 19 May @ 18:30: Well, Jessica Lee of RunBare has replied to my email already (I love it when people are prompt repliers):
From Jessica Lee To Colin Seymour Date Wed, May 19, 2010 at 5:42 PM Subject RE: Purchasing from the UK Hi Colin, I'm sorry for your troubles. Unfortunately, our shipping costs to the UK are exorbitantly high $15 US + the handling costs from our fulfillment house. We are currently working with the main distributor of Vibram FiveFingers in the UK to also distribute the book to UK customers. Hopefully we'll have a cost effective solution for you soon. We'll let you know as soon as we do. Best regards, Jessica Lee, President RunBare™
So when I know more, I'll let you all know.
Update - 9 Jul 2010 @ 11:45: Looks like Jessica forgot about me as I've just discovered the book is now available to buy in the UK.


>
Sem Ⅳ
Assignments For Combinatorics and Graph Theory
Solving problems on string,sets and binomail coefficients.
Solving problem using mathematical induction
Solving problem on Eulerian and Hamiltonian graph
Solving problem Prims Algorithm
Solving Problem on Kruskal's Algorithm
Solving problem on Dijkstra's Algorithm
Solving problem on finding Augmenting path in network flows
Solving problem on network flow using Ford and Fulkeson's labeling algorithm
Solving problem on chromatic number and colouring


am an aspiring programmer who wants to combine my knowledge of scientific principles and coding to make the science industry better equiped to
handle the ever changing world. As previously said I took sciences in high school and that is where I found my passion
In my life I have been fortunate enough to study in three different countries. here are all the schools:
This is my first time coding. In high school at St. Christopher I took IT as a class and passed it every year I took it. The class was easy so
I thought I woud one day want to take something similar again. AS a programmer in training at Moringa School I have learnt the basics of:
This is a link to the project I've worked on in my time at Moringa here.


1- Search
Search on Madrassa is simple. Type your prefered school in Dubai & you will see all you need to know!!
Search on Madrassa is simple. Type your prefered school in Dubai & you will see all you need to know!!
Madrassa provides perfect location to earn your future educational goal. Locating a school is just one-click!!
We simplify every step in the application!
It is now easy to apply to any school in Dubai.
Now that you've applied, wait for your enrollment in 24hrs!!
That's all....
Find a school by using our filter search engine
We make school information easy to find in Dubai
It has never been easy but Madrassa did it!!


Each year we work with several hundred organizations to help them overcome challenges they’re facing and increase their social impact. We’ve worked with organizations as large as Red Cross and World Vision, right through to small start-up social enterprises.
We help organizations to...
Being selected to receive consulting services is a competitive process. Typically less than 30% of organizations that apply for consulting services receive it.
In order to be selected as a 180 Degrees Consultant, applicants need to (a) have a very strong academic record, (b) prove relevant experience and/or ability, and (c) go through one or more interviews to ensure that their motivations and values are consistent with those of 180 Degrees Consulting.
Our consultants receive both general and project-specific training from world leading top-tier consulting companies. Many of our 180 Degrees Consultants also received mentoring from full-time consultants with decades of professional experience.
180 U of T is one of 88 branches internationally. 180 Degrees Consulting has had engagements with international non-profits such as Red Cross and World Vision. What makes 180 Degrees Consulting different is that we are able to utilize resources from our international network to make a difference in the local communities.
On consultant team can consist of a Masters student at the Munk School of Global Affairs, an Industrial Engineering undergraduate student, and a Marketing student from the Rotman School of Management. The diversity of our consultants allows us to specialize for any particular engagement.
Each consulting project is led by a Team Leader, who is overseen by a Branch Consulting Director, who is then in turn managed by an International Consulting Director. This three-tier quality control process ensures our work is always of the highest standard.
When you recieve consulting services from 180 UofT, you also recieve consulting services from resources at the University of Toronto. Our consultants utilize (a) advisement from the expert faculty at the University of Toronto, and (b) as students we have access to particular resources provided from the school.
Even though we have a high-performing executive team at 180 UofT, all branch-wide decision making is reviewed and rigorously thought through with our expert board of advisors consisting of community leaders and faculty members.
The alumni of 180 Degrees Consulting - University of Toronto go on to work for top-tier companies, successfully launch start-ups, and attend graduate school in Ivy League universities. We have access to the guidance and expertise of our overachieving alumni.
Every consultant, committee member, and executive member of 180 Degrees Consulting is here on a volunteer-basis. The reason we commit our early morning weekends and light nights on mondays to the work is because each member believes they are making a difference. Toronto is our home and we strive to make our mark and see the legacy we leave behind.
We are always accepting new requests for our consulting services. We are currently reviewing our engagement wait lists and time lines in the near future. Click on the right to link to our head form, or feel free to email<EMAIL_ADDRESS>for an immediate response.


This is my TODO list
"new" - Add new Item to List
"list" - View existing list
"delete" - remove a specific element
"quit" - Quit list application
Execute startList() in console to start the todo list


Site is currently down due to API being shut down
Last Updated: {{ app.date | date: "dd-MMM-yyyy, hh:mm a" | uppercase }}
This map is ran by BNO News, however I am working on a new map that will be out soon! Thank you for waiting
Coronavirus disease (COVID-19) is an infectious disease caused by a new virus. The disease causes respiratory illness (like the flu) with symptoms such as a cough, fever, and in more severe cases, difficulty breathing. You can protect yourself by washing your hands frequently, avoiding touching your face, and avoiding close contact (1 meter or 3 feet) with people who are unwell.


Load
(Positive) Thick Lens in Air
(Negative) Thick lens in Air
Thin lens
Single thin Lenses
Multiple thin lenses
Gullstrand Eye Model (Relaxed)
Gullstrand Eye Model (Accommodated)
Empty
Get Report
Lens Prescription
Objects and Images
Summary
Add Element
Index
Sphere
Thick
Thin
Delete Selected
Clear
Reset
Add Light
Object
Image
Delete Selected
Clear
Reset
Summary Information goes here!
Add Lens Element
Add Object or Image


This is a 30 Second begins with a person walking through a wooded area in the rain, there is the distant sound of footsteps through the different terrain which grow louder as the person moves to a more sheltered space. The person then arrives at a building and enters.
For the video project, after the public screening i decided to try and fix the obivious audio problems, such as the dry sound from the voice recording sessions, also the problems with the voices not syncing properly to the audio. I linked the project over from premiere pro to Audition where i first focused on the voice sync problem and i was able to fix most of this using the voice synchronization tool on the problematic clips. from the original vidio audio, i was able to reference the pitch that was made by the actors on the day of shooting. the other tools i used were the noise remover, dehisser, the parametric equalizer, the hard edge limiter and the 20 band equalizer. this removed most of the bad high and low frequincies but it was still hard to remove some of the nasally sounds from the voices.


Hi,
I am currently pursuing Masters in Software Engineering at San Jose State University, California. I did undergraduation from University of Mumbai with a Bechelor's degree in Computer Engineering. My primary areas of interest include Software Development, Distributed Computing, Data Analysis and Data Mining.
I am proficient in Java, JavaScript, Python, NodeJS and C# and have worked with C before. I enjoy developing web applications and I am skilled in HTML5, CSS, JavaScript and frameworks like Bootstrap. Additionally, I have experience developing backends for multiple companies.
In my spare time I like to take online courses and learn new things. I am a design addict at heart who believes in 'less is more' ideology. I manage to squeeze in time to keep up with latest development in tech and build rapid prototypes. Delicious Food is what I live for.


I'm an honors student at Drexel University majoring in Computer Science. It has always been my dream to work as a senior developer on a huge project that will change the world. I've done a lot in preparation to achieve this goal, and I'm finally ready for real world experience.
• Code and maintain website using HTML, Bootstrap, JavaScript, and CSS
• Develop backend algorithms in Python to optimize the development process
• Produce a website with a working JavaScript contact form using Bootstrap and HTML
• Use Basecamp/Slack to communicate project due dates and team roles
• Work as a member of a team to produce a mobile app that automates CRM software
• Program in Unity and C# to develop a multiplayer desktop game
• Work with a team following the agile development method
• Follow a Gantt chart to complete tasks by a deadline
• Worked as a cashier and then promoted to head chef
• Trained new employees for both cashier and cook positions
• Developed fully functional website with HTML, JQuery, CSS, and JavaScript that displayed the products of a local businesses
• Used GitHub as a source management tool to work on a project with multiple people
Examples:
GPA: 3.84
GPA: 3.9 (Unweighted)
GPA: 5.1 (Weighted)
Apart from being a developer, I enjoy most of my time being active. I like to watch YouTube videos and learn as much as possible. During the warmer months, I enjoy going to the beach and relaxing.
When forced indoors, I watch a variety of TV shows, ranging from Game of Thrones to Chopped Junior.


| ${value} Second Duration | 100 |
| ${value}m Radius | 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 |
| ${value} Burn Damage over ${duration} Second(s) | min: [65.0, 80.0, 95.0, 110.0, 125.0, 140.0, 160.0, 180.0, 205.0, 230.0] Burn Damage over min: 1.0 Second(s) |
| ${value}% Physical Resistance | -33.6 -42 -50.4 -58.8 -67.2 -75.6 -84 -92.4 -100.8 -109.2 |
| MaxLevel | 6 |
| UltimateLevel | 10 |


To Do List
Input Task To Do:
SUBMIT
Tasks remaining: 2
Task No.1:
This is task number one
Completed
Delete
Task No.2:
This is task number two
Completed
Delete
Completed tasks:
✔ Task 3
✔ Task 4
✔ Task 5
✔ Task 6


| Growth | Income | Growth+Income | |
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| No Protection | {{ $ctrl.invGoal["7"] }} results | {{ $ctrl.invGoal["8"] }} results | {{ $ctrl.invGoal["9"] }} results |
| Name | Company | Gender | Protection | Goal | |
|---|---|---|---|---|---|
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History of The Internet
Home
Our Team
About
Invention of the Internet
Browser Wars
Dot Com Bubble
People
Internet Search
Timeline
Welcome to the History of the Internet Website!


Photo caption Photo caption Photo caption Photo caption Photo caption Photo caption Photo caption Photo caption Photo caption


Arizona mother arrested after accidentally shooting 13-year-old son, police say
by Talia KaplanA 13-year-old boy was seriously injured on Saturday after his mother accidentally shot him during a fight outside an Arizona home, police said.
Phoenix police said the shooting unfolded in the backyard of a home on East Taylor Street around 5:20 p.m. on Saturday.
They said the 13-year-old was in the backyard when a man named Michael Wilson, 59, confronted an unwanted guest outside, which led to a fight.
Police said the teen’s mother, Tonya Monroe, 41, went to get a gun from their home to break up the fight and accidentally fired it, striking her son.
The 13-year-old was taken to a nearby hospital with serious injuries. Doctors later listed him in stable condition, according to police.
NEW MEXICO HUNTER SHOT BY DOG SAYS DOG WAS FATALLY SHOT WEEKS LATER: REPORT
Police arrested both Wilson and Monroe. The teen’s mother faced several charges including aggravated assault and disorderly conduct involving a weapon.
Wilson was accused of drug and weapons possession. The unwanted guest, who did not report any injuries, did not cooperate with investigators, police said.
CLICK HERE FOR THE FOX NEWS APP
Investigators said they developed probable cause to arrest Monroe and Wilson based on interviews and evidence found inside the home.
The investigation was ongoing.


--- layout: none ---
I am a Canadian citizen, but worked in the San Francisco Bay Area from 2005-2012. I am currently limiting my entries into the USA as I need to spend 365 days outside the country to reset my H-1B visa status.
I will be working on personal projects and traveling until at least April 2013.
Based in Vancouver, Canada
website
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{% email<EMAIL_ADDRESS>email %}
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LinkedIn
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GitHub
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Ohloh
Help people to cooperate at internet scale.
The Wikimedia Foundation is the organization behind Wikipedia. I worked on HTML5 interfaces, multimedia, file storage, internationalization, APIs, GUI editing, parsing, live collaboration while editing, and more (commit log). Frequently deployed and configured code running live on all Wikipedia. Mentored other paid and community developers, including Google Summer of Code 2011, and winning team for Wikipedia SF Hackathon in January 2012. Left amicably to pursue personal projects and reset visa status.
Flickr is one of the leading photo-sharing websites in the world. I joined the PHP team and made various improvements to Flickr's payment system, FlickrMail, and mobile APIs. I was let go as part of Yahoo-wide layoffs in April 2009.
Upcoming.org is a collaborative events calendar, acquired by Yahoo in 2005, using PHP and Perl. I integrated event listings into Yahoo Search, redesigned the event data and geographic models, improved user interface and responsiveness, wrote a deploy system to automatically optimize site load time, and mentored new hires. Received Excellence and Hack Day Awards in 2008.
I was part of the team that launched Google Checkout, a Java-based commerce and payments system. My primary role was to refactor Google Checkout for international markets. This included the frontend, the data store, order processing and taxation policy, the Merchant API and more. I also worked closely with the Google Accounts team to create a Checkout-specific account creation flow.
Enfold Systems provides consulting and enterprise tools for Plone, an open source, Python-based content management system. I left the company amicably when I got a job offer from Google.
Lickr was the first complete AJAX interface for Flickr, implemented as a Greasemonkey extension for Firefox. Previously, Flickr had relied on Flash and it was an open question whether an AJAX interface was even possible or desirable. Flickr followed soon after with their own AJAX interface for all browsers.
During this period I consulted for a number of clients, particularly Vision2Hire Solutions (acquired by BrainHunter), a maker of online human resources software.
ActiveState produces tools for scripting language programmers. I re-engineered Programmer's Package Manager Server, part of a system for installing Perl and Python software libraries. I also helped architect and code the ActiveState Programmer Network, a site which aggregated knowledge about such languages.
At Stylus, I designed and programmed web applications for some of Canada's most successful corporations.
Bachelor of Arts, Communication Studies, Spring 2000
Concordia University, Montreal, Quebec
English, French
Open Source, OSS, Javascript, Node.JS, jQuery, JavaScript, PHP, Python, Perl, Java, MediaWiki, JasmineBDD, PHPUnit, JUnit, XML, HTTP, HTML, HTML5, SMTP, REST, Apache, MySQL, Linux, GLaDOS, UX, User Experience
Available upon {% email<EMAIL_ADDRESS>request %} . Also see LinkedIn.


--- layout: post title: "The Week in Predictions" date: 2018-01-06 09:50:00 +0000 tags: image: https://s3.amazonaws.com/contrafabulists/2018-01-06-owl.jpg imagecredits: https://www.flickr.com/photos/petermgriffin/7257604030 ---
The Contrafabulists – Kin Lane and Audrey Watters – monitor predictions that technologists and marketers make about the future of technology. No surprise, most of the predictions are made about AI, a development whose future folks have been predicting incorrectly since the 1950s.
Here are the predictions made this week:
From Paleofuture: “Predictions for the Year 2018 From the 20th Century.”
“Some 2018 Predictions” from Washington State University’s Mike Caulfield.
Via eSchool News: “25 education trends for 2018.”
According to Edsurge, “Measuring Learning Will Be Key to Improving It in 2018.”
Folks really really really want AR and VR to be a thing – this year as in the past. According to MIT Technology Review, these are “Five of the Coolest-Looking VR and AR Headsets, Apps, and More Coming in 2018.”
Via Futurism: “California Is Officially Planning to Ban Polluting Cars by 2040.”
Via Fortune: “Amazon Will Buy Target in 2018, Tech Analyst Predicts.” From Business Insider: “There is a 40% chance Apple will acquire Netflix, according to Citi.”
“Does Life In 2018 Live up to What We Predicted a Century Ago?” asks Futurism.