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We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.
Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 µm) method that captures the pathogen's spores (zoospores) and is later used to amplify the pathogen's DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.
Recycling water in farms and nurseries is becoming increasingly popular due to the increase in water costs and environmental concerns behind water usage. Many irrigation methods have been developed for growers to reduce the spread and occurrence of plant disease. Regardless of the source of the water (irrigation or precipitation), runoff is generated, and many vegetable and nursery growers have a pond to collect and recycle runoff1. This creates a reservoir for possible pathogen accumulation favoring the spread of pathogens when the recycled water is used to irrigate crops2,3,4. Oomycete plant pathogens particularly benefit from this practice as zoospores will accumulate in water and the primary dispersive spore is self-motile but requires surface water5,6,7. Phytophthora capsici is an oomycete pathogen that affects a significant number of solanaceous and cucurbit crops in different ways8. Often, the symptoms are damping-off of seedlings, root and crown rot; however, in crops such as cucumber, squash, melon, pumpkin, watermelon, eggplant and pepper, entire harvests may be lost due to fruit rot9. Although there are known methods of detecting this plant pathogen, most require an infection to have already taken place which is too late for any preventative fungicides to have a significant effect10.
The traditional method to test irrigation water for the detection and diagnosis of targeted microorganisms is an antiquated approach when speed and sensitivity are crucial to success and profitable crop production11,12. Plant tissue susceptible to the targeted pathogen (e.g., eggplant for P. capsici) is attached to a modified trap that is suspended in an irrigation pond for extended period before being removed and inspected for infection. Samples from the plant tissue are then plated on semi-selective media (PARPH) and incubated for culture growth, then morphological identification is performed using a compound microscope13. There are other similar detection methods for other plant pathogens using selective media and plating small amounts of contaminated water before sub-culturing14,15. These methods require anywhere from 2 to 6 weeks, several rounds of sub-culturing to isolate the organism, and experience on Phytophthora diagnostics to be able to recognize the key morphological characters of each species. These traditional methods do not work well for detection of irrigation water contaminated by P. capsici due to factors such as interference by other microorganisms that are also present in the water sources. Some fast-growing microorganisms like Pythium spp. and water-borne bacteria can overgrow on the plate making P. capsici undetectable16,17.
The purpose of this study was to develop a sensitive and specific molecular method that can be used in both field and laboratory settings to detect P. capsici zoospores in irrigation water. The protocol includes the development of a novel loop-mediated isothermal amplification (LAMP) primer set able to specifically amplify P. capsici, based on a 1121-base pair (bp) fragment of P. capsici18,19. A previously developed LAMP primer from Dong et al. (2015) was used in comparison to the assay that was developed for this study20.
The LAMP assay is a relatively new form of molecular detection that has been demonstrated to be more rapid, sensitive, and specific than conventional polymerase chain reaction (PCR)21. In general, conventional PCR assays cannot detect under 500 copies (1.25 pg/µL); in contrast, previous studies have shown that the sensitivity of LAMP can be 10 to 1,000 times higher than conventional PCR and can easily detect even 1 fg/µL of genomic DNA22,23. Additionally, the assay can be carried out rapidly (often in 30 min) and on-site (in the field) by using a portable heating block for amplification and a colorimetric dye that changes color for a positive sample (removing the need for electrophoresis). In this study, we compared the sensitivity of PCR and LAMP assays using a filter extraction method. The proposed detection method allows researchers and extension agents to easily detect the presence of P. capsici spores from different water sources in less than two hours. The assay is proven to be more sensitive than conventional PCR and was validated in situ by detecting the presence of the pathogen in the irrigation water used by a grower. This detection method will allow growers to estimate the presence and population density of the pathogen in various water sources that are being used for irrigation, preventing devastating outbreaks and economic losses.
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1. On-site detection of Phytophthora capsici from irrigation water using portable loop-mediated isothermal amplification
- Setting up the pump and filter
- Attach a filtering flask to a tube that is connected to a hand pump so that when the pump is activated, air will be pulled in through the mouth of the filtering flask.
- Fit the Buchner funnel into the rubber stopper to the mouth of the filtering flask and fit the appropriately sized piece of filter paper into the Buchner funnel so that air is pulled through the filter paper. The filter paper should have a retention size of 15 µm.
NOTE: The filter paper must fit to the edges of the Buchner funnel so that minimal water will flow around the filter paper.
- Water sampling and filtering
- Take water samples from the targeted source. Water may have small amounts of debris but not significant sediment or soil.
- Pour up to 1,000 mL (1 Liter) of test water over the filter paper placed inside the Buchner funnel slowly enough to prevent overflow, while the hand pump (or vacuum) is being used to create a suction to pull the water through.
NOTE: There is no minimum amount of water that can be tested using this method, and although it at least 50 mL is suggested, 1000 mL is the maximum for this method.
- Using forceps, remove the filter paper from the Buchner funnel and cut it into small pieces with sterile scissors. Add as many pieces (8-12) as can be submerged in the amount of extraction buffer (400 µL for magnetic bead-based extraction) required by the protocol into a 1.5 mL tube. Save remaining pieces of filter paper for processing after the first set has been extracted.
- Vortex or otherwise agitate the pieces of filter paper and extraction buffer for 10 s every minute for 5 min. Then, using forceps, remove the filter paper losing as little of the extraction buffer as possible. Repeat this step with the remaining pieces of filter paper until all pieces have been vortexed/agitated and soaked in the extraction buffer.
- Magnetic bead-based extraction of DNA from filter paper
- To the 1.5 mL tube (which now contains approximately 200-300 µL of extraction buffer), add 20 µL of proteinase K and 10 µL of 10 ng/µL RNase.
- Incubate at room temperature for 15 min, vortex or shake the tube every 3 min.
- Add 500 µL of magnetic beads with the binding buffer to the sample and mix well by shaking. Then incubate for 5 min at room temperature.
- Place the tube in the magnetic separator rack for 2 min until all beads have been pulled to the magnet. Remove and discard the supernatant.
- Remove the tube from the magnetic separator. Add 500 µL of Wash Buffer 1 and re-suspend beads by shaking the tube vigorously. Wait for 30 s and then place the tube back in the magnetic separator. Wait for 2 min until all beads have been pulled towards the magnet before removing and discarding the supernatant.
NOTE: When waiting for the magnetic beads to magnetize to the separator, we recommend inverting the tube, which can dislodge magnetic beads stuck to the cap and the sides of the tube and result in a greater number of beads being attached.
- Repeat step 1.3.5 with 500 µL of Wash Buffer 2.
- Repeat step 1.3.5 with 500 µL of 80% ethanol.
- Airdry the magnetic bead pellet for 15 min at room temperature (18-27 °C) with the lid open. If temperatures do not permit, incubate in a gloved hand with the cap open for 15 min.
- Remove the tube from the magnetic separator. Add 50 µL of elution buffer and re-suspend the beads by pipetting up and down for 1 min.
- Place tube back in a magnetic separator. Wait 2 min before transferring the supernatant without disturbing the beads to a separate tube for DNA storage.
NOTE: Here the experiment can be paused before moving on. Extracted DNA should be stored on ice or in a -20 °C freezer.
- Application of newly developed LAMP assay
- Prepare LAMP primer mix using 0.2 µM of each F3 and B3 primer, 0.8 µM of each Loop-F and Loop-B primer, and 1.6 µM of each FIP and BIP primer (Table 2).
- Add the following LAMP solution to a single PCR tube, or to each individual tube in the 8 tube strip: 2.5 µL of primer mix (step 1.4.1), 12.5 µL of LAVA LAMP master mix, 1 µL of extracted DNA, and 9 µL of ddH2O. The total volume is 25 µL.
NOTE: If using the portable amplification instrument (e.g., Genie III) with colorimetric dye (e.g., Warmstart), refer to section 2.4.2- 2.4.3.
- Designate two tubes as positive and negative controls. For the positive control, use either a control provided by the LAVA LAMP kit, or a known positive DNA sample. For the negative control, use ddH2O. For both, substitute the 1 µL of extracted DNA for 1 µL of the positive control or ddH2O.
- Set the samples into a heat block (or portable amplification instrument) set to 64 °C for 45 min.
NOTE: Other extraction methods can be used here instead of magnetic bead based extraction. CTAB and a commercial DNA extraction kit were both successfully tested using the standard protocols and substituting the filter paper pieces for the plant sample24,25. Results were compared in Table 2. If concentration of DNA can be quantified, use between 1-10 ng genomic DNA.
- Visualization of results
- If performed in a laboratory setting, view the amplification products by loading 5 µL of each sample into a 1% agarose gel, running them in a gel electrophoresis machine, and imaging them in a UV imaging machine.
- If a colorimetric dye (e.g., Warmstart) was used, view the color change to determine results as positive or negative.
- If a portable amplification instrument (e.g., Genie III) was used, view the amplification graph on the screen to determine the results.
- Application of previously developed PCR assay
NOTE: If using a conventional PCR assay, steps 1-3 remain the same, and the following steps should be applied in place of steps 1.4 and 1.5.
- Add 1 µL of each DNA extraction to individual tubes containing the following components: 12.5 µL of Green PCR master mix, 9.5 µL of ddH2O, and 1 µL of forward and reverse primers (Table 2).
- Spin down each sample using a microcentrifuge and place tubes into a thermal cycler.
- Use the following thermal cycler settings in accordance to the previous publications: 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min.
- Run the product in a 1% agarose gel. Observe the presence of bands under UV light where positive reactions will have a band size of ~508 bp.
- Traditional method of detection
NOTE: There are multiple methods of selective plating for pathogen detection, and the following is a general protocol for P. capsici.
- First, obtain a healthy eggplant fruit (a susceptible host for P. capsici) and surface sterilize by washing the fruit surface with 70% isopropyl alcohol.
- Place the eggplant fruit into milk crates with a flotation device (polyethylene foam or other) and deploy into irrigation ponds. Secure each bait trap to a single point and leave the traps in the water reservoir (recycled irrigation water) for at least 7 days or until fruit rot symptoms are observed. Collect the fruit and transport it to the laboratory.
- Rinse and dry the fruit in a sterile hood before removing small pieces of infected tissues and place them on a plate of PARPH medium amended with 25 mg/L pentachloronitrobenzene, 0.0005% pimaricin, 250 mg/L ampicillin, 10 mg/L rifampicin and 50 mg/L hymexazol. Incubate plates at 25 °C for 5 days.
- View plates under a compound microscope for traditional morphological identification at 4 days after isolation.
2. Determining the detection limit of zoospore concentration
- Making zoospore suspension
- Incubate P. capsici on V8 agar (100 mL of V8 juice, 900 mL of ddH2O, 1 g of CaCO3) plates for 1 week at 26 °C. Multiple plates can be used to obtain a larger amount of zoospore suspension.
- Incubate the plates under continuous light at room temperature for 3 days to stimulate sporulation.
- Flood the plates by adding 15 mL of ddH2O to each plate and place them in a 4 °C fridge for 25 min. Then return to room temperature for 30 min.
- Agitate plates to dislodge zoospores and pipette the solution into a single 50 mL tube from all the plates.
- To obtain an accurate estimate of zoospore concentration, add 10 µL of the spore suspension onto a hemocytometer and observe under a microscope to count zoospores and estimate the average concentration.
- Serial dilution
- Add 1 mL of the spore suspension and 9 mL of ddH2O to a separate tube. Repeat this step for as many 10-fold dilutions as desired.
- Spore solutions are then submitted for DNA extraction based on the previous protocol using the filtration method.
NOTE: If a larger volume of suspension is desired, double the volume: 2 mL of spore suspension and 18 mL of ddH2O.
- Detection of zoospore concentration limit
- Evaluate spore detection limit by running each serial dilution individually through the assay until clearly positive results are no longer observed. Once the final dilution is obtained, dilute by a factor of 2 (4.8 to 2.4 in this example) and run the assay again to get a more accurate detection limit.
- Development and optimization of the LAMP method
NOTE: LAMP primers were designed based on a 1121-base pair (bp) fragment of P. capsici (Li et al.19) as shown in Supplementary Figure 2.
- If colorimetric dye (e.g., Warmstart) is used, use the following the solution: 2.5 µL of primer mix, 12.5 µL of colorimetric dye, 0.5 µL of Green fluorescent dye, 1 µL of extracted DNA, and 8.5 µL of ddH2O. The total volume is 25 µL.
- When using the portable amplification instrument with LAVALAMP mastermix, have an initial step of 95 °C for 3 min as recommended by the manufacturer, but this is not required. A final annealing step is not required to observe the color change or amplification graph. Do not run a warmstart step if the commercial colorimetric dye is to be used.
- View LAMP assay results in one of the following methods: run samples on a 1% agarose gel or view using a UV imaging machine with the naked eye or view at the Genie III real-time amplification screen.
- Optimize the temperature of the LAMP assay by using the portable amplification instrument and analyzed using the real-time amplification graph for speed and level of sensitivity. Run samples with unique temperatures to determine the fastest amplification with the highest level of sensitivity.
- Determine the detection limit of the LAMP developed assay by making a serial dilution of extracted DNA (as with spore suspension in step 2.2.1) and maintaining reaction conditions as previously described for the LAMP reaction for each dilution.
- Detection limit determination and comparison with conventional PCR method
- Use DNA extracted in steps in section 1.3 to compare the detection level of conventional PCR with that of the LAMP assay.
- Add 1 µL of DNA to a PCR tube that contained 1 µL of both forward and reverse PCR primers (Table 2), 12.5 µL of Green PCR Mastermix, and 9.5 µL of ddH2O for a total of 25 µL.
- Amplify samples in a thermal cycler using the following conditions: 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min.
- Run samples in an electrophoresis machine on a 1% agarose gel and view on a UV imaging machine. The excepted band size was 508 bp.
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Representative Results
Optimization of LAMP method
In this study, we detected the presence of Phytophthora capsici in irrigation water using a portable loop-mediated isothermal amplification (LAMP) assay. First, the proposed LAMP assay was optimized by testing different LAMP primer concentrations [F3, B3 (0.1–0.5 µM each); LF, LB (0.5–1.0 µM each) and FIP, BIP (0.8–2.4 µM each)], durations (30–70 min), and temperatures (55–70 °C). The final LAMP primer mix used in this study was: 0.2 µM of each F3 and B3 primer, 0.8 µM of each Loop-F and Loop-B primer, 1.6 µM of each FIP and BIP primer. Optimization of reaction temperature was performed in the portable amplification instrument (e.g., Genie III) by determining what temperature performed the fastest reaction with no additional negative amplification. The optimal temperature was confirmed to be 64 °C (data not shown). The optimal time for running the assay at 64 °C was 45 min, as the lowest concentrations that were positive for detection (1.2 x 102 spores/mL) still amplified by 40 min, while higher concentrations amplified at 20 min (Figure 2D). The amplified LAMP products were further observed on 1% agarose gel stained with a nucleic acid stain to confirm amplification. All reactions were repeated at least three times.
Isolates of P. capsici were taken from Tennessee, Florida, and Georgia and were submitted to the same protocol described in the methods. All samples of P. capsici isolates were amplified successfully in all runs of the assay (Figure 3).
Detection and sensitivity testing of Phytophthora capsici in irrigation water using portable LAMP assay
We standardized this filter paper-based LAMP method under laboratory conditions using a serial dilution of P. capsici spore suspensions (Figure 1). Serial dilutions were made from a P. capsici spore suspension starting at 4.8 x 104 zoospores/mL and run with the LAMP assay in triplicate. Spore concentrations are shown rather than DNA concentration due to the method involved for DNA extraction. CTAB DNA extraction of the highest spore concentration was 4.5 ng/µL measured by Nanodrop, and the magnetic bead DNA extraction protocol yielded 3.8 ng/µL26. The newly designed LAMP primer set could detect a concentration as low as 1.2 x 102 spores/mL (Figure 2B, 2C, & 2D) with all methods of extraction. The sensitivity shown in the graph of amplification on the portable amplification instrument was identical to that shown in the UV image with no additional sensitivity. The same serial dilution was run in a LAMP reaction using the colorimetric dye to determine the level of sensitivity to the naked eye for field detection. The lowest observable concentration was 4.8 x 103 spores/mL (Figure 2C).
To evaluate the ability of this assay using real-world samples, water samples were collected from seven ponds used for commercial vegetable production in Tift County, Georgia (Table 1, Figure 5, and Supplementary Figure 1). Out of the 7 ponds, 3 showed positive LAMP results (P1, P4, and P6) (Figure 6A, 6B, & 6C). These results suggest that the portable filter paper-based LAMP method could be very useful for detection of the pathogen even with a low zoospore concentration. This demonstrates the applicability of LAMP as a more sensitive detection assay than PCR for screening irrigation water contamination by P. capsici.
Comparative analysis of different methods: Traditional baiting, conventional PCR, and the portable LAMP-based assay
In order to compare the detection sensitivity of LAMP with conventional PCR, the DNA extracted from the serial dilution of spore suspensions were run in a PCR reaction. Results showed that conventional PCR was 40x less sensitive than LAMP, only able to detect a zoospore concentration as low as 4.8 x 103 spores/mL (Figure 2A). Additionally, the DNA samples obtained from filtered irrigation pond water were tested using conventional PCR, and only one of the three positive samples (P4) was successfully amplified as expected band size plus significant contaminates resulting in some smearing and unspecific bands (Figure 6D). Table 3 displays the differences between detection methods using such variables as time, cost, sensitivity, and preparation required. LAMP was the least expensive method among these three methods and it was also the fastest, ranging from 30-60 min for amplification (DNA extraction excluded). Conventional PCR ranged from 120-180 min for amplification (DNA extraction excluded).
Finally, to determine the specificity of the primers, samples of closely related oomycete pathogens (Phytophthora sansomeana, Phytophthora sojae, Phytophthora cinnamomi, Phytophthora palmivora, Pythium ultimum var. ultimum, Phytopythium vexans, Phytopythium helicoides, and Pythium aphanidermatum) were obtained, DNA was extracted using the same protocol for consistency and evaluated by the new LAMP assay with a positive and negative control (Figure 4) to determine specificity of the primers. All non-target samples were negative using the optimized 64 °C for 45 min. This was observed on the real-time amplification graph and imaged on a 1% agarose gel under UV light.
Figure 1: Diagram showing the different steps involved in Phytophthora capsici from a serial dilution of a concentrated spore suspension under laboratory conditions. Please click here to view a larger version of this figure.
Figure 2: Laboratory optimization of the limit for detection of Phytophthora capsici. (A) Conventional PCR assay was carried out using specific P. capsici primers on serial dilution factors and visualized on 1% agarose gel. 1, Ladder; 2-7, 4.8 x 104, 4.8 x 103, 4.8 x 102, 2.4 x 102, 1.2 x 102 spores/mL, respectively and 7, negative water control. (B) LAMP assay serial dilution factors visualized on 1% agarose gel. 1-6, a decreasing spore concentration: 4.8 x 104, 4.8 x 103, 4.8 x 102, 2.4 x 102, 1.2 x 102 spores/mL, and 7, negative water control. (C) LAMP results visualized using the colorimetric dye. 1-6, a decreasing spore concentration: 4.8 x 104, 4.8 x 103, 4.8 x 102, 2.4 x 102, 1.2 x 102 spores/mL, and 7, negative water control. (D) LAMP results visualized on the amplification graph. Red = 4.8 x 104, Dark blue = 4.8 x 103, Orange = 4.8 x 102, Light blue = 2.4 x 102, Green = 1.2 x 102 spores/mL, Pink = Negative control (other Phytophthora species), Yellow = ddH2O. (E) A standard curve showing quantification of the values shown in the real-time results. Ln (Spore count) is shown on the X-axis, and minutes to amplification on the Y-axis. (F) LAMP assay with published primer (Dong et al. 2015) on serial dilution factors visualized on 1% agarose gel. 1-6, a decreasing spore concentration: 4.8 x 104, 4.8 x 103, 4.8 x 102, 2.4 x 102, 1.2 x 102 spores/mL, and 7, negative water control. Please click here to view a larger version of this figure.
Figure 3: Amplification of P. capsici DNA from various locations. (A) LAMP results visualized on 1% agarose gel. 1, PC_TN1; 2, PC_TN2; 3, PC_FL1; 4,PC_FL2; 5, PC_GA1; 6, PC_GA2; N, Negative control. Samples 1 and 2 were isolated from TN; samples 3 and 4 isolated from FL; samples 5 and 6 were isolated from GA. (B) Results visualized using the colorimetric dye. 1, PC_TN1; 2, PCTN2; 3, PC_FL1; 4,PC_FL2; 5, PC_GA1; 6, PC_GA2. (C) Results visualized on the amplification graph. Red, PC_TN1; Orange, PCTN2; Yellow, PC_FL1; Green, PC_FL2; Dark blue, PC_GA1; Light blue, PC_GA2; Pink, negative control. Please click here to view a larger version of this figure.
Figure 4: Specificity determination of LAMP assay using DNA from non-target species P. capsici. (A) LAMP assay reaction with related non-target species on agarose gel and visualized on 1% agarose gel. L, Ladder; 1, Phytophthora sansomeana; 2, Phytophthora sojae; 3, Phytophthora cinnamomi; 4, Phytophthora palmivora; 5, Pythium ultimum var. ultimum; 6, Phytopythium vexans; 7, Negative control; 8, Phytophthora capsica. (B) LAMP results visualized using the colorimetric dye. 1, Phytophthora sansomeana; 2, Phytophthora sojae; 3, Phytophthora cinnamomi; 4, Phytophthora palmivora; 5, Pythium ultimum var. ultimum; 6, Phytopythium vexans; 7, Negative control; 8, Phytophthora capsici (C) LAMP results visualized on the amplification graph. Red = Phytophthora capcisi, all other non-target species samples were not amplified. Please click here to view a larger version of this figure.
Figure 5: Pictures showing the sampling and processing of recycled water for the detection of Phytophthora capsici in the field. Please click here to view a larger version of this figure.
Figure 6: Results from on-site detection of Phytophthora capsici in irrigation water sources. (A) Agarose gel showing results from the LAMP amplification of tested water from seven farm in South Georgia. Sample names from left to right: P1, P2, P3, P4, P5, P6, P7, Negative control, N. (B) LAMP results visualized using warmstart colorimetric dye of field samples: P1, P2, P3, P4, P5, P6, P7, Negative control, N. (C) Results from LAMP amplification of field samples using graph. Red: P1, Green: P2, Purple: P3, Yellow: P4, Blue: P5, Orange: P6, Pink: P7, Negative control, N. (D) Agarose gel showing conventional PCR results of the amplification using specific primers PC-1/PC-2 (note than only one site tested positive in comparison to three in LAMP). Please click here to view a larger version of this figure.
|Pond name
|County, State
|Target crops for irrigation
|PCR Detection
|LAMP Detection
|History of Disease (Y/N)
|Tift, GA
|Tift, GA
|Tift, GA
|Tift, GA
|Tift, GA
|Tift, GA
|Tift, GA
Table 1: Detection of irrigation water from Southern GA.
|Primer type
|Primer name
|Sequence 5'-3'
|This study
|This study
|This study
|This study
|This study
|This study
|Zhang et al., 2006
|Zhang et al., 2006
Table 2: Primers used in this study.
|Conventional PCR
|4.8 X 102 spores/ml
|1.2 X 102 spores/ml
|2 weeks or longer
|2-3 hours (not including DNA extraction)
|30 mins - 1 hour (not including DNA extraction)
|• Media creation
• Plating and isolation and
• Designing a trap
|• Spore collection using Filter paper
• DNA extraction and
• PCR assay
|• Spore collection using Filter paper
• DNA extraction and
• LAMP assay
|• Autoclave
• Media and plates
• Incubation room
• Flow hood
• Eggplant
• Milk Crate
|• Thermal cycler
• Agar gel
• Gel Doc
• Heat block or
• Genie III
|$5.00 per trap
|$0.60 per reaction
|$0.75 per reaction
Table 3: Comparison of methods for detection of P. capsici.
Supplementary Figure 1: Location of Tift County, GA and samples of irrigation water taken from various locations from within the state. Positive samples were shown as red dots. Please click here to download this figure.
Supplementary Figure 2: Design of LAMP primer set. Arrows show direction of how primers are read. Please click here to download this figure.
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The testing of irrigation water for phytopathogens is a crucial step for growers using irrigation ponds and recycled water27. Irrigation ponds provide a reservoir and breeding ground for a number of phytopathogens as excess irrigation water is directed from the field to the pond carrying with it any pathogens that may have been present16,27. The traditional method for detection of a plant pathogens in a large water source is to set a bait for the pathogen by using susceptible host tissue (e.g., fruit, leaves) suspended in the pond and wait for an infection to take place, then remove the fruit/leaves and confirm the diagnosis with microscopy or molecular methods13,14. These methods are limiting due to the amount of time required to run the detection test (2 weeks or longer), and the labor and equipment required. Additionally, extensive experience and knowledge in visual diagnosis, pathogen morphology, and taxonomy are required for accurate results. Molecular techniques such as PCR, qPCR, and DNA hybridization require significantly less time (3-4 h) than the traditional methods of detection; however, they require expensive equipment and a laboratory setting. Additionally, these techniques do not allow for the processing of large volumes of water. Serological assays too, fall short in their detection ability due to non-target positive reactions, and no species-specific assays for Phytophthora species have been developed. Loop-mediated isothermal amplification (LAMP) has recently been used as an on-site diagnosis technique for rapid and sensitive detection of multiple pathogens as the assay only requires a single temperature rather than a thermal cycler28,29. LAMP can be run in the field using a colorimetric dye for visual confirmation or using a real-time amplification machine for results in less than one hour30.
The goal of this experiment was to develop a rapid and sensitive method to detect the presence of Phytophthora capsici in water sources either on-site or in a laboratory. To increase the speed of detection and to combat the limitations of the previously mentioned methods for detecting P. capsici in irrigation water, we designed a method using filter paper to capture the spores and extract their DNA from a larger volume of water. After spores were captured using the filter paper technique and DNA was extracted, the presence of the pathogen was confirmed based on a newly designed LAMP primer set specific to P. capsici. Detection sensitivity and specificity was compared using LAMP and PCR. In all 3 replications and with all of the zoospore concentrations, LAMP was a quicker and more sensitive detection method (Table 3). This method is not limited by having a small sample volume as traditional methods, as this method can be test up to 1 L of water at a single time, increasing the chances of pathogen detection. It was noted in testing that pouring irrigation water slowly through the Buchner funnel at a speed of no more than 40 mL per second increased the spore capture ability of the filter paper.
To validate the detection protocol, water samples from the field where P. capsici was suspected to be present were also taken (Supplementary Figure 1) to test the designed method with a practical scenario. Out of the 7 farms tested, 3 were positive for the presence of P. capsici using the LAMP assay (Figure 6A-6C) while only one farm was positive when using the conventional PCR assay (Figure 6D), showing LAMP as a more sensitive assay for this method of testing irrigation water. Although, this filter based LAMP assay could detect DNA from a concentration as low as 1.2 x 102 zoospores/mL which was significantly less than the original sensitivity of this assay (0.01 ng genomic DNA equivalent to ~5 spores) with unfiltered zoospore suspension. The previous LAMP assay by Dong et. al. (2015)20 was run on the same serial dilution and the level of sensitivity was the same (Figure 2F). The level of detection for a PCR assay with unfiltered spore solution have also shown a higher level of detection (equivalent ~10 spores) which was very similar to the previous PCR based findings10. The spore detection limit of the traditional baiting method of detection was not checked as a single spore could cause infection depending on its individual ability. The decreased sensitivity found in both LAMP and PCR assays is likely due to some spores flowing through or around the filter paper, or once attached to the filter paper, unable to be extracted with 100% efficiency. Nevertheless, this new LAMP and filter system can process and analyze a much higher volume of water than previous methods and confers a higher level of specificity and speed for in-field detection.
Of the extraction methods used, magnetic bead-based extraction was the most rapid and did not require the use of external machines such as a bead beater or centrifuge making it useful for in-field extractions and compliments the portable feature of the LAMP assay. The CTAB based method yielded the highest concentration of DNA but took the longest amount of time, while the commercial plant DNA extraction kit (e.g., DNeasy) was second in both time required and DNA concentration acquired.
With both CTAB and the commercial plant DNA extraction kit, during the homogenization of filter paper it was noted that homogenization was more successful and yielded a higher concentration if the CTAB solution (or extraction buffer) was added to the tube with the pieces of filter paper before bead beating or hand homogenization commenced. Bead beating was done 3 times for one minute each, but vortexing and agitating the tube in between each round was necessary for complete homogenization in all extraction methods. Importantly, the filter paper is subject to getting stuck on the sides or bottom of the tube, so it is crucial to make sure the filter paper is off the walls so that it gets homogenized.
The total time required for amplification is 90-120 min and can be easily done in the field for on-site diagnosis. This filter method is also designed to filter significant amounts of water to increase the possible chances for the detection of the pathogen. This method is also applicable to many pathogens that can accumulate in a water source, particularly genera such as: Pythium, Phytophthora, Fusarium, and bacteria; the only change required will be the development of an equally specific LAMP primer set for the targeted pathogen30.
A significant output of this work is the development of a highly sensitive and rapid filter paper-based LAMP assay for the detection of P. capsici in irrigation water sources. We expect that this study will lead to an increase in awareness of contamination of recycled irrigation water, eventually improving management of Phytophthora associated diseases, and consequently reduce production costs and increase crop yield. Such information is highly needed to improve vegetable production sustainability and enhance profitability of vegetable productions.
Subscription Required. Please recommend JoVE to your librarian.
The authors have nothing to disclose or any conflicts of interest.
This work received the financial support of Georgia Commodity Commission for Vegetables project ID# FP00016659. The authors thank Dr. Pingsheng Ji, University of Georgia and Dr. Anne Dorrance, Ohio State University for providing pure cultures of Phytophthora spp. We also thank Li Wang and Deloris Veney for their technical assistance throughout the study.
|Catalog Number
|Agarose gel powder
|Thomas Scientific
|Buchner funnel
|Southern Labware
|Bullet Blender
|Next Advance
|Centrifuge 5430
|Fischer Scientific
|CTAB solution
|Dneasy Extraction Kit
|Filter Flask
|Filter Paper
|United Scientific Supplies
|Gel Green 10000X
|Thomas Scientific
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|UV Gel Doc
|Analytik Jena
|Warmstart Colorimetric Dye
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|70% isopropanol
|Fischer Scientific
- Hong, C., Moorman, G. J. Plant pathogens in irrigation water: challenges and opportunities. Critical Reviews in Plant Sciences. 24, (3), 189-208 (2005).
- Malkawi, H. I., Mohammad, M. J. Physiology, Genetics, Morphology, & Microorganisms, E. o. Survival and accumulation of microorganisms in soils irrigated with secondary treated wastewater. Journal of Basic Microbiology. 43, (1), 47-55 (2003).
- Bush, E. A., Hong, C., Stromberg, E. L. Fluctuations of Phytophthora and Pythium spp. in components of a recycling irrigation system. Plant Disease. 87, (12), 1500-1506 (2003).
- Ghimire, S. R., et al. Distribution and diversity of Phytophthora species in nursery irrigation reservoir adopting water recycling system during winter months. Journal of Phytopathology. 159, (11-12), 713-719 (2011).
- Hausbeck, M. K., Lamour, K. H. Phytophthora capsici on vegetable crops: research progress and management challenges. Plant Disease. 88, (12), 1292-1303 (2004).
- Gevens, A., Donahoo, R., Lamour, K., Hausbeck, M. Characterization of Phytophthora capsici from Michigan surface irrigation water. Phytopathology. 97, (4), 421-428 (2007).
- Thomson, S., Allen, R. Occurrence of Phytophthora species and other potential plant pathogens in recycled irrigation water. Plant Disease Reporter. 58, (10), 945-949 (1974).
- Lamour, K. H., Stam, R., Jupe, J., Huitema, E. The oomycete broad-host-range pathogen Phytophthora capsici. Journal of Molecular Plant Pathology. 13, (4), 329-337 (2012).
- Sanogo, S., Ji, P. Water management in relation to control of Phytophthora capsici in vegetable crops. Agricultural Water Management. 129, 113-119 (2013).
- Zhang, Z., Li, Y., Fan, H., Wang, Y., Zheng, X. Molecular detection of Phytophthora capsici in infected plant tissues, soil and water. Plant Pathology. 55, (6), 770-775 (2006).
- Trout, C., Ristaino, J., Madritch, M., Wangsomboondee, T. Rapid detection of Phytophthora infestans in late blight-infected potato and tomato using PCR. Plant Disease. 81, (9), 1042-1048 (1997).
- Sankaran, S., Mishra, A., Ehsani, R., Davis, C. A review of advanced techniques for detecting plant diseases. Commputers and Electronics in Agriculture. 72, (1), 1-13 (2010).
- Wang, Z., et al. Development of an improved isolation approach and simple sequence repeat markers to characterize Phytophthora capsici populations in irrigation ponds in southern Georgia. Applied and Environmental Microbiology. 75, (17), 5467-5473 (2009).
- Ali-Shtayeh, M., MacDonald, J. Occurrence of Phytophthora species in irrigation water in the Nablus area (West Bank of Jordan). Phytopathologia Mediterranea. 143-150 (1991).
- Pringsh, P. Comparison of serological and culture plate methods for detecting species of Phytophthora, Pythium, and Rhizoctonia in ornamental plants. Plant Disease. 74, (9), 655 (1990).
- Stewart-Wade, S. M. Plant pathogens in recycled irrigation water in commercial plant nurseries and greenhouses: their detection and management. Irrigation Science. 29, (4), 267-297 (2011).
- Aragaki, M., Uchida, J. Y. Morphological distinctions between Phytophthora capsici and P. tropicalis sp. nov. Mycologia. 93, (1), 137-145 (2001).
- Tomlinson, J., Boonham, N. Potential of LAMP for detection of plant pathogens. CAB Reviews Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources. 3, (066), 1-7 (2008).
- Li, P., et al. A PCR-based assay for distinguishing between A1 and A2 mating types of Phytophthora capsici. Journal of the American Society for Horticultural Science. 142, (4), 260-264 (2017).
- Dong, Z., et al. Loop-mediated isothermal amplification assay for sensitive and rapid detection of Phytophthora capsici. Canadian Journal of Plant Pathology. 37, (4), 485-494 (2015).
- Khan, M., et al. Comparative evaluation of the LAMP assay and PCR-based assays for the rapid detection of Alternaria solani. Frontiers in Microbiology. 9, 2089 (2018).
- Sowmya, N., Thakur, M., Manonmani, H. K. Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods. Journal of Applied Microbiology. 113, (1), 106-113 (2012).
- Waliullah, S., et al. Comparative analysis of different molecular and serological methods for detection of Xylella fastidiosa in blueberry. PLOS ONE. 14, (9), 0221903 (2019).
- Böhm, J., et al. Real-time quantitative PCR: DNA determination in isolated spores of the mycorrhizal fungus Glomus mosseae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants. Journal of Phytopathology. 147, 409-416 (1999).
- Klimczak, L., Prell, H. J. C. Isolation and characterization of mitochondrial DNA of the oomycetous fungus Phytophthora infestans. Current Genetics. 8, (4), 323-326 (1984).
- Ghimire, S. R., et al. Detection of Phytophthora species in a run-off water retention basin at a commercial nursery in plant hardiness zones 7 b of Virginia in winter. Phytopathology. 96, (6), (2006).
- Feng, W., Hieno, A., Kusunoki, M., Suga, H., Kageyama, K. J. P. LAMP detection of four plant-pathogenic oomycetes and its application in lettuce fields. Plant Disease. 103, (2), 298-307 (2019).
- Aglietti, C., et al. Real-time loop-mediated isothermal amplification: an early-warning tool for quarantine plant pathogen detection. AMB Express. 9, (1), 50 (2019).
- Almasi, M. A. Development of a colorimetric loop-mediated isothermal amplification assay for the visual detection of Fusarium oxysporum f. sp. melonis. Horticultural Plant Journal. 5, (3), 129-136 (2019).
- Gill, D. J. Pathogenic Pythium from irrigation ponds. Plant Disease Reporter. 54, (12), 1077-1079 (1970).


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DETROIT (AP) -- The U.S. Justice Department says a Detroit-area woman has pleaded guilty to conspiracy on accusations of trying to steal $15.9 million from the Medicare program for bogus mental health care.
The government said Wednesday that 63-year-old Louisa Thompson made the plea Wednesday. She faces up to 10 years in prison and a $250,000 fine at her sentencing Oct. 18 before Detroit U.S. District Court Judge Nancy Edmunds.
The Justice Department says Thompson began making fraudulent Medicare billings in 2006 through TGW Medical Inc. and Caldwell Thompson Manor Inc. The government says she billed for psychotherapy that was either not performed or given by unqualified providers.
The government says Medicare paid about $4.9 million on the claims.
Published: Fri, Jun 22, 2012


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Every day is Christmas for a dog surrounded with love. Perfectly plush, this adorable Bichon Frisé makes a wonderful Christmas companion. A wonderful gift for the puppy lover in your life
Product Features:
Tan Bichon with red reindeer antlers
Shiny red jingle bell on a black and white checkered collar
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Material(s): polyester/foam
- Plush Bichon Frisé dog Christmas figure
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Another fantastic indie title to add to the growing collection on Nintendo Switch!
Category: News
For the second time in a row arguably the best was saved till last in a Nintendo Direct. Link is dancing into an indie game!
We've finally had more details on the two games coming at the end of the year for Nintendo Switch. Pokemon Sword and Shield!
So we've finally had the first Nintendo Direct of the year. What a direct it was! Whilst there were some noticeable omissions, namely Metroid Prime Trilogy and a Nintendo Switch Online update, the direct was cram packed. It was nearly 40 minutes of non stop switch games.
We've finally had the announcement from that the first Nintendo Direct of 2019 will be tomorrow at 10pm UK time (2pm PT & 5PM ET).
February's Nintendo Switch Online NES Games have been announced. The two games that have been announced will be released on the 13th of this month, so not long too wait till you get your hands on them.
Links to 2019 gaming events
Gamescom is one of the worlds largest gaming events held every year in Cologne, Germany. Last year the event attracted exhibitors from over 56 countries. Around 370,000 visitors attended gamescom 2018.
E3 is arguable the biggest gaming event on the calendar. It is held in Los Angeles in California in the US once a year usually around the middle of June.


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This scope has been mounted on a .22-250 since new. One very minor ring mark forward of turrets - barely visible. Otherwise scope & glass are in excellant shape. Gloss finish. A.O. focuses down to 25 yards. Duplex reticle. I do not have original box, but will ship in a padded scope box. Will include sunshade shown in photo's. $435 shipped/insured in lower 48. Will go by 1st I will take it timestamp as it is listed on multiple sites.
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There are 10 new articles in PLoS ONE this week. As always, you should rate the articles, post notes and comments and send trackbacks when you blog about the papers. Here are my own picks for the week - you go and look for your own favourites:
Targeted Destruction of Photosensitive Retinal Ganglion Cells with a Saporin Conjugate Alters the Effects of Light on Mouse Circadian Rhythms:
Non-image related responses to light, such as the synchronization of circadian rhythms to the day/night cycle, are mediated by classical rod/cone photoreceptors and by a small subset of retinal ganglion cells that are intrinsically photosensitive, expressing the photopigment, melanopsin. This raises the possibility that the melanopsin cells may be serving as a conduit for photic information detected by the rods and/or cones. To test this idea, we developed a specific immunotoxin consisting of an anti-melanopsin antibody conjugated to the ribosome-inactivating protein, saporin. Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells. We find that the specific loss of these cells in the adult mouse retina alters the effects of light on circadian rhythms. In particular, the photosensitivity of the circadian system is significantly attenuated. A subset of animals becomes non-responsive to the light/dark cycle, a characteristic previously observed in mice lacking rods, cones, and functional melanopsin cells. Mice lacking melanopsin cells are also unable to show light induced negative masking, a phenomenon known to be mediated by such cells, but both visual cliff and light/dark preference responses are normal. These data suggest that cells containing melanopsin do indeed function as a conduit for rod and/or cone information for certain non-image forming visual responses. Furthermore, we have developed a technique to specifically ablate melanopsin cells in the fully developed adult retina. This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies.
Toward a Comprehensive Approach to the Collection and Analysis of Pica Substances, with Emphasis on Geophagic Materials:
Pica, the craving and subsequent consumption of non-food substances such as earth, charcoal, and raw starch, has been an enigma for more than 2000 years. Currently, there are little available data for testing major hypotheses about pica because of methodological limitations and lack of attention to the problem. In this paper we critically review procedures and guidelines for interviews and sample collection that are appropriate for a wide variety of pica substances. In addition, we outline methodologies for the physical, mineralogical, and chemical characterization of these substances, with particular focus on geophagic soils and clays. Many of these methods are standard procedures in anthropological, soil, or nutritional sciences, but have rarely or never been applied to the study of pica. Physical properties of geophagic materials including color, particle size distribution, consistency and dispersion/flocculation (coagulation) should be assessed by appropriate methods. Quantitative mineralogical analyses by X-ray diffraction should be made on bulk material as well as on separated clay fractions, and the various clay minerals should be characterized by a variety of supplementary tests. Concentrations of minerals should be determined using X-ray fluorescence for non-food substances and inductively coupled plasma-atomic emission spectroscopy for food-like substances. pH, salt content, cation exchange capacity, organic carbon content and labile forms of iron oxide should also be determined. Finally, analyses relating to biological interactions are recommended, including determination of the bioavailability of nutrients and other bioactive components from pica substances, as well as their detoxification capacities and parasitological profiles. This is the first review of appropriate methodologies for the study of human pica. The comprehensive and multi-disciplinary approach to the collection and analysis of pica substances detailed here is a necessary preliminary step to understanding the nutritional enigma of non-food consumption.
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2009 Fiat 500 LOUNGE 3-Door
Engine & MPG
- Engine Size:1242cc
- MPG Extra-urban:65.7mpg
- MPG Combined:55.4mpg
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Dimensions & Weight
- Height:1,488 mm
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Fiat 500 Additional Information
FULL SERVICE HISTORY, JUST BEEN SERVICED, GREAT CONDITION BOTH INSIDE & OUT, BLUETOOTH/MP3/AUX, CHEAP INSURANCE & TAX, 50+ MPG, 12 MONTHS MOT, 3 MONTH WARRANTY, 3 MONTH AA COVER, 3 MONTH BREAKDOWN COVER & 2 KEYS. Here at Kent County Cars we are a small family run business with over 25 years in the motor trade. All vehicles are hand picked from various sources we have acquired over this time and are all HPI clear so buying can be done with confidence. It is our primary aim to offer quality used cars to the public at a competitive price. Unlike many others we pride our self on customer service before, during and after you acquire your new vehicle from us, meaning no query is too big or small. All vehicles are sold with 12 months MOT, 3 months AA cover and up to 5 years AA warranty. We are also pleased to announce that we can now offer same day £0 deposit finance at very competitive rates with flexible adjustable monthly payments to suit each customer individually. We are open from 9.00am - 7.30pm 7 days a week. Viewing by appointment only.
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Watch Orphan Black Season 2 Episode 3 online and prepare to meet Cal. He's Kira's father!
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Watch Orphan Black Season 2 Episode 3 online and get to know Cal, an ex-boyfriend of Sarah's.
Episode Details
Sarah reunites with an ex-boyfriend on this episode of Orphan Black. She also realizes that she can't just run away from her problems.
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- Season:
- Orphan Black Season 2
- Episode Number:
- 3


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Thursday, September 6, 2012 6:29pm ET by Newsdesk
Jay-Z wants daughter Blue Ivy to "love herself" and "know who she is"
Music mogul Jay-Z has opened up about his 9-month-old daughter, Blue Ivy, and exclaimed "my family's amazing"
In a new interview with The New York Times Style Magazine, the '99 Problems' hitmaker spoke about his own upbringing and explained that he doesn't want his baby to go through what he did:
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Watch Jay-Z's video for 'Otis' below:
Latest 10 JAY Z News Stories


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Wenjing Zhao* and Feng Shi Pages 128 - 134 ( 7 )
Background: Accurate identification lymph nodes in multi-slice CT images enables promptly diagnosing and correctly treating of cancers and subsequent measuring the effect of the treatment. Computer-aided detection (CAD) systems are necessary choice to reduce labor intensity of radiologists and to do the work with higher accuracy than the artificial recognition. The detection of lymph node is non-trivial since the lymph nodes vary in shape and there is not significant contrast to their surrounding regions, which makes the effect of the classifiers based on features of either boundaries or shapes of the lymph nodes unsatisfactory. Recently, the feature extraction from intra lymph nodes gets more attention than those from the borders and the shapes.
Method: In the paper, the lymph node was segmented by a Random Forest model. 500 random contextual features were extracted for each voxel of the lymph node. In order to improve performance, we proposed the scaling features in a Random Forest classifier without any extra complexity.
Result: We testified our method on 10 mediastinum lymph nodes from TCIA (the Cancer Imaging Archive) database. We improved the performance of the random forest model by the scaled features. After we adjusted the model parameters and chose for features with high information gains, our Random Forest classifier reached better performance.
Conclusion: A simpler, faster and more efficient method is searched for enabling practicable computeraided diagnosis and computer-aided detection in the field of the lymph node segmentation. Since the scaling could ensure equal treatment of the features with different absolute value in the classifier, the precision and the recall of our Random Forest classifier were increased based on the scaled features.
Boundary detection, data scaling, image segmentation, mediastinal lymph node, object detection, random forest.
College of Sciences, Northeastern University, Shenyang, College of Computer Science and Engineering, Northeastern University, Shenyang


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Daya Brought the Energy to THON 2020
Which holiday weekend in State College is your favorite? Some may say State Patty's weekend, or some will even say Arts Fest weekend. However, for…Read More
Which holiday weekend in State College is your favorite? Some may say State Patty's weekend, or some will even say Arts Fest weekend. However, for…Read More
When you're not dancing away in the BJC this weekend, it's vital that you make the most out of resting to refuel your body. Help…Read More
THON is the biggest way Penn State gives back, but it's far from the only. Now that a successful THON weekend has passed, there are still plenty of opportunities for service on campus.Read More
When it comes to THON weekend, the Bryce Jordan Center is jammed pack with all sorts of crazy costumes, neon colors and diy accessories as far as the eye can see. In the sea of THON dancers, VALLEY set out to find some of most creative looks of THON 2019.Read More
This weekend, two of the 708 Penn Staters dancing their hearts out are also members of VALLEY! We're extremely proud of our editor-in-chief, Tess Petak,…Read More
Since 1973, Penn State students have participated in the year-long fundraising efforts that benefit Four Diamonds at Penn State Children's Hospital. What started as an…Read More
Practically the whole school is busy preparing for one of the most major events of the year, and the members of VALLEY are no exception…Read More
So you're a committee member for THON 2018? First of all, know you're about to have one of the most amazing weekends of your life….Read More
Around 7:30 p.m. in the BJC, THON participants started to get antsy. There was nothing officially scheduled for the night, but many students had been…Read More
Here at VALLEY we know that THON weekend can be one of the most amazing, unforgettable, but tiring experiences for any Penn State student. We've…Read More


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I did a rocket launch today. I think it was pretty fun. I was nervous that my rocket might blow up, but I put six layers of tape to connect the nosecone with the body. In the beginning, we had to do research. I didn't like it. It was boring to me. But I did like making the inspiration boards. My team made a logo with our team name, the New Newtons, on it. We put pictures and information that we thought would help us on the launch on our board. A few days before, we had to prepare and learn to use the tools that we'll be using on the launch day. One of them was the clinometer. The clinometer looked like a protractor with an arrow, a button, and a handle. Mastering the clinometer was hard. I was afraid that I would fail to use the clinometer rightly when the launch day came. There was also another tool: the trundle wheel. The trundle wheel measured the distance between the clinometer readers and the rocket. It was very resourceful. The preparation was tricky, but fun. I had three jobs, but only one for my launch. I was the countdown person for my launch. I was really happy to have that job. But I also was a clinometer reader for the group 1,2,3 Blastoff! and was a data recorder for the group the 3 Engineers. After the launch, I really wanted to do it again. I never wanted to stop. My teacher said that it would take an hour minimum for all the rockets to launch, but instead, it took forty five minutes. I think my group worked really well. We didn't fight. I had to keep on checking my clipboard to see when I was up. And after I did my first job, I almost forgot that my group's rocket was going to launch, and nearly missed the launch. But good thing I checked my clipboard. It was very useful. We were allowed to bring our iPhones outside. Some people did. I recorded some of the launches for two people. I observed that even though our rocket was the smallest rocket, we went higher than a taller rocket. So I don't think that the height of the rocket mattered. But we had eight fins, and I think that helped. I don't think our rocket needs changes. Maybe more tape?
Tag Archives: Science
Plant Blog Post #2
For our Controlled quad, our tallest plant is about 11 centimeters tall. The leaves and stem is green and the flower is yellow. The most flowers we have are 3. At the beginning, our tallest was probably 2 centimeters tall. Our plants grew about 9 centimeters tall throughout the weeks. I think that the yellow flower is a Buttercup or Sunflower. Some of our plants are going to snap soon.
For our Manipulated quad, cell 1, 2 and 4 is dead. Only cell 3 is surviving. I think this happened because we have really little soil in the Manipulated quad. The tallest measurement for that cell is 13 centimeters tall. It has a tiny flower that is yellow and the stem and leaves are green. In the beginning, the tallest was about 6 centimeters tall. Our plants grew about 7 centimeters tall throughout the weeks.
The outcome are a few yellow flowers. The yellow flowers bloomed from the buds. I think this outcome happened because of the seeds, water and sunlight. Also, photosynthesis.
Plant Blog Post #1
My class and I are learning about plants in our science unit we're doing right now. We learned a lot. I am with a group of three girls, including me. Our scientific question for this experiment is: What is the effect of having less soil on plant growth? Our hypothesis is: If we put less soil in the cells, then the plant won't grow as high everyday because the plant doesn't have enough soil. Our controlled quad is normal. We are manipulating the soil.
We have two quads. One quad is controlled and one quad is manipulated. Controlled means that everything is the same. Manipulated is where we change one thing in the process. We have four cells in each quad. Everytime we water the cells, we took note of what was happening. For our Controlled quad, the tallest plant is 2 centimeters tall. The color of the plant is green. Our Manipulated quad's plants are 3 centimeters taller. What we changed in our Manipulated quad was the amount of soil. It turned out to be taller than I thought.
Maglev Post #2
What we did today that worked was that we got the magnets on the track to line up with the magnets on the train. Before we figured out that we had to do that, our magnets on our track attracted with the magnets on the train. We needed like poles and since the magnets are not labeled, we needed to find a system. So we marked the tape on the magnets with stars so we would know which side would repel and which side would attract. We also got the train to levitate a little higher with some more strip magnets.
What we did today that failed was that we tried to put some magnets on the side the train was going to on the attract side and a few magnets on the beginning of the track on the repel side. We thought the magnets on the repel side would give a push to the train and the train would get attracted to the magnets on the attract side so the train would move there, but that didn't work. I learned from my failure that we needed stronger magnetic forces.
My goals for the next session is to get the train to move to the other side with a slight push and for the train to not tip over.
Maglev Train Post #1
A maglev train is a train that works on magnets. It levitates and goes really fast. The maglev train levitates because of magnets. The magnets on the track repels with the magnets on the train. My group's idea was to put 4-6 strip magnets on the track and 1-2 disc magnets on the train.
Today, we made our train levitate. Before, we got it to levitate but then one of our group members wanted to tape the magnet on the train and after she did that, we couldn't make it levitate again. But we couldn't make the maglev train move with a slight push.
The challenging part was to make the train levitate, then it was making the train move with a slight push. What worked well was that my group got to make our train levitate on the train track. Also, we all worked together so that was the best part. I think this activity is really fun.


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Seasalt Carved Wood Dress - Harbour
This Carved Wood Dress is made from cool, breathable linen garment-dyed for incredible softness and a sun-faded hue. Inspired by '40s style shirtwaisters, this midi length style is cleverly tailored for a flattering fit with a full skirt for a feminine, vintage feel and a deep waistband with under bust pleats. It's finished with a low V-neck, short sleeves and fabric covered buttons.
- 100% Linen
- A-line
- Midi-length
- Short sleeves
- V-neck
- Garment-dyed
- Wrap-around patch pockets
- Fabric covered buttons
- Button front
- Machine wash at 30 degrees
Standard Delivery
£3.75 or Free for orders over £100
(UK only)
Please allow 2-5 working days for delivery
Express Delivery
£4.95 for delivery within 3 days
(UK only)
Place your order by 12pm for express service
Click & Collect
Place your order on & collect from our store in Whitstable
Please allow 1 day before collection of order
International Delivery
£12 for European Delivery with a minimum order value of £30.
Please allow 5-14 working days for delivery
We hope you and your purchases will be very happy together!
If not, return your purchases within 14 days for an exchange or refund
Contact Us
Visit us at 39 Harbour Street, CT5 1HS
Call us on 01227 275900
Email us on email@example.com


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custom floral letter wood christmas ornament | floral initial | laser engraved | CHOOSE YOUR LETTER
+ One laser engraved wood ornament (approx. 3.5" round)
+ Red ribbon
+ Choose your letter in the dropdown box
Gift boxing is not available.
Since this is a handmade item, variations and textures are normal. This adds character and each piece is unique in itself!
TURNAROUND TIME: 5-7 business days


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An East Midlands Ambulance worker is amongst thousands of British citizens who are stranded in the world's largest coronavirus lockdown in India.
As a key NHS front line worker, Suman Legah is eager to get back to the UK, to serve her community in this time of need during the COVID 19 panemic.
Suman arrived in India with four family members on March 13, expecting to enjoy a short 10 day holiday for a temple festival and to attend a wedding.
However the NHS worker's trip has taken a dramatic turn as the Indian police attended their relative's home in Goraya City, Punjab on March 22.
Suman told ITV Central that when the police arrived they immediately wanted to know where her and her family had come from and then proceeded to check each of their temperatures.
Without any notice the household was issued with a coronavirus quarantine notice which stated that none of the residents of the house were allowed to leave the vicinity until 6th April.
The lockdown date has now been extended to April 14 after the Indian government announced a 21-day lockdown for the entire country on March 24.
Suman and her family members are becoming increasingly anxious as food and water supplies are starting to run low.
"Food and water is running low and this is day 6 of being indoors. The police are roaming streets and dispersing crowds of more than 2 people together and beating them with truncheons. My family can't go out in fear they will beat us too, drag us away like they are doing as it's shown on local tv here."
The Foreign and Commonwealth Office told us they understand British tourists are finding it hard to return home, and they're working round the clock to try to help.
They say the government is trying to keep key routes open as long as possible, and consular staff are giving advice to those still abroad.
Suman and other British people who are stranded in India have been keeping each other motivated during these challenging times by commenting on a Facebook group called 'Brits Stranded in India'. So far the group has over 800 followers.
Some members of the group have taken to twitter to seek help from the Government.
A friend of Suman's has also started an online petition called 'Repatriate UK citizens stuck in India'.
The Foreign and Commonwealth Office (FCO) has told ITV Central that their teams are working around the clock to make sure all British people who need help are receiving support.
"We recognize British tourists abroad are finding it difficult to return to the UK because of the unprecedented international travel and domestic restrictions that are being introduced around the world – often with very little or no notice.
The FCO has also issued the following advice:
You should read and follow the FCO travel advice and the advice of the local authorities where you are.
You should contact your airline, tour operator and travel insurer to find out what support they can offer.
The FCO provides information on what to do and who to contact if you require financial assistance abroad.
We also have online guidance for British people travelling and living overseas following the outbreak of coronavirus.
- Full FCO Travel Advice for India can be found here - https://www.gov.uk/foreign-travel-advice/india.
If any British national in India requires immediate consular assistance, they should contact FCO on New Delhi: +91 (11) 2419 2100; Goa: +91 832 6636800; Chennai: + 91 (44) 42192151; Mumbai:+91 (22) 6650 2222.


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Get the Link to This Publication
You can copy and paste this link into any site, or bookmark it online or offline with any service.
- Done
Digital Includes: PDF WebViewer
- Details
- Description
- Published by:
- Gianna Nicole
- Published:
- 9/3/2017
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Standard / 8.25" x 10.75"148 pages Perfect-bound
- Category:
- Fashion & Style
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NYFW Magazine is dedicated to the Emerging Artist. Designers, Models, Actors, Dancers, Photographers, and the rest of the arts community.


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ALL >> Health >> View Article
Herbal Insomnia Supplements To Treat Sleeplessness
When one is feeling trouble in sleeping despite being extremely tired, then he/she is definitely suffering from insomnia. It is a condition in which one struggles hard to sleep or unintentionally wakes up at night and stays awake for long time for no reason. Using tranquilizers to get sound sleep makes one dependent on such pills. The person suffering from insomnia does not feel refreshed even after sleeping for hours. This causes dizziness at daytime which distracts him/her from focusing on work.
This dizziness makes their mind unconscious and forces to sleep even if the brain knows you are doing something important. Stress and improper sleep may permanently damage brain functions and swipe of memory. Along with these factors, deficiency of nutrients too affects sleep.
With changing time, sleep habits have also changed. Most of the people use cell phones, tablets, laptops in bed. Some people work late on computer at night or watch television before going to bed. Such irregular routine of sleeping creates problems in sleeping and one may pass time by just changing positions. Eating heavy meals and drinking beverages at dinner time keeps one awake even if you are tired. Nearby environment factors also disturbs your sleep like small kids who often wake up at night, person snoring near you, high noise in surroundings, etc. Excessive work out at night time also keeps body and brain active for relatively long time and does not allow taking sleep.
Herbal supplements are the best for such health issues as they provide natural treatment to overcome insomnia problem. With Aaram capsules, which are herbal insomnia supplements, one gets complete rest both physically and mentally. These capsules not only make brain unconscious but also relax muscles and reduce tiredness naturally. This relaxed state of mind helps in falling into sleep easily. Herbal formula of these supplements provides nourishment to brain cells. This increases ability of brain to think, understand and judge a condition. This lets brain to judge when to stay awake or sleep. These herbal capsules also suppress the effects of other diseases like jaundice, diabetes, etc., on mind which badly affects the functioning of brain and causes trouble in sleeping.
Herbal supplements to treat sleeplessness not only solve sleep problems but also enhance brain functions. On getting proper rest, brain power and memory increases. While sleeping, our mind recollects memories and thus people who sleep properly have better memory. This is why kids have better learning power because they sleep more in comparison to adults. These herbal insomnia supplements reduce the rush of hyperactive and negative thoughts. Aaram capsules especially help elders who easily become victim of forgetfulness habit.
Using Aaram capsules regularly reduces the risk of serious problems like sleep walking and mental instability. Both men and women can use these capsules without any risk of side effects. These herbal insomnia supplements give positive results within just 3-4 months. These capsules can be used for prolong time also as these do not cause any adverse effects on health. It is also suggested to avoid heavy meals at night and develop routine of getting to bed on time daily.
Read about Best Ayurvedic Treatments For Insomnia. Also know Ayurvedic Herbal Treatment Pills For Insomnia. Read about Sleeplessness Herbal Pills.
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You're 64. You are returning from a trip that has you bringing home vodka as a souvenir.
What would possess you to chug that two pints of vodka? What possible explanation could there be for such behavior?
- A gun to the head?
- A tightening vice to the scrotum?
- Being forced to watch a 12-hour Rosie O'Donnell marathon?
What about a pesky airport fee for checking a carry-on bag?
"… he was told at a security check that he would have to either throw out the bottle of vodka or pay a fee to have his carry-on bag checked. Instead, he chugged the vodka, and was quickly unable to stand or otherwise function, police said."
Well… yeah.
That would have to be one heck of an airport fee to make me risk getting extremely ill and spending a few days in the hospital. (Putting the risk of death aside for a moment of course… )
How do you get to be 64 years old and not realize that chugging two pints of vodka is going to f&#* you up? Anyone?


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Tag Archives: otaku
The war of baseball
After watching whole lots of baseball anime from the old Princess 9 (Thats a female baseball anime,but still…) to the recent Ookiku Furikabutte,my friend and I decided to look for a batting cage to put our skills to the test,and … Continue reading
Posted in Otaku events in Malaysia Tagged anime, baseball, otaku 1 Comment
Tribute to my Nendroid Azu-nyan
Just got my nendroid Azunyan,or also be called Azuza from K-On last week and got so attracted to it and decided to make a comic strip on her…Hope that you all like it… A little notice to everyone also that … Continue reading
Posted in Kuso Manga Strip, Otaku's room photos Tagged Azuza, figures, K-On, Nendroid, otaku Leave a comment
Why Otaku stays single….
Ever wonder why a real otaku always single?Well…thats because you are not full otaku unless you do the below… Who says the legendary everything mix up curry only available in Anime? Otaku may be single but will never lack companion … Continue reading
Posted in Otaku's room photos Tagged otaku, Otaku room 2 Comments
My New Collection-Black Rock Shooter Blade ver.
After months of waiting after the 1st version of Black Rock Shooter by Good Smile Company was first released (which I apparently failed to get…),the 2nd version which is black blade mode has finally made its appearance.As a fan of … Continue reading
Posted in Otaku's room photos Tagged Black Rock Shooter, figures, Good Smile Company, otaku Leave a comment
Gundam Challenge 2010
It happens every year but the impressive works of the Gundam modification have never ceased to impress me.Although the participating works tend to decrease from last year,the quality of the creation have improved which makes up the lack of amount.Kudos … Continue reading
Posted in Otaku events in Malaysia Tagged Gundam, malaysia, otaku, Otaku events Leave a comment
An Otariman's room exposed!
For my 2nd post I would like to give everyone a sneak peak on my room itself.Well, even though it probably wouldn't be much to show compared to a heavy duty Otaku who might have their room filled with figures,but … Continue reading
Posted in Otaku's room photos Tagged fate stay night, figurines, Ironman, LuckyStar, malaysia, otaku, otariman, saber Leave a comment
Me & Otaku culture-an intro of why I started this blog
Hi everyone and welcome to my blog!Since this is my 1st article,is better that I introduce myself myself…I am Haoh Kishin, an English translation of the kanji word 霸王机神(read it in Japanese and is how is pronounce…)The name is an … Continue reading
Posted in Intro Tagged anime, hokuto shin ken, malaysia, otaku Leave a comment


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A. R. Frederiksen
YA/A Fantasy Author
She woke, not feeling her heart.
"If there ever again was a day like this, flowers in her hair and wind in her ears, she would not be fooled twice. Not by him, and not by herself. But, of course, what else could she ever do?"


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Choose yourself. A small voice tells you otherwise. Force in the thousand voices it commands. Shouting at you. Faceless. Cowardice. Victims too of the game.
Change. This heat from within excites. Pleases. An awakening spiritual. Physical. Knowing who you are. Who you have always been. Filled. Unmasked your destiny a reflection of the soul untethered. This heart beats strong. Desire heavy in your loins. Wanton. Simple need lay heavy upon your breast. Kiss these lips. Taste the woman. Unchained explore her soft curves. Mind of steel. Touch her. She soars. Her wings kissing the wind. A miracle. Her enlightenment.


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Peer reviewed
Direct link
Direct link
ERIC Number: EJ969586
Record Type: Journal
Publication Date: 2012
Pages: 8
Abstractor: ERIC
Stem Cells, Science, and Public Reasoning
Hurlbut, J. Benjamin; Robert, Jason Scott
Journal of Policy Analysis and Management, v31 n3 p707-714 Sum 2012
These are interesting days in the scientific, social, and political debates about human embryonic stem cell research. Pluripotent stem cells--cells that can, in principle, give rise to the body's full range of cell types--were previously derivable only from human embryos that were destroyed in the process. Now, a variety of somatic cell types can be reprogrammed to a pluripotent state. Meanwhile, long-standing promises about the therapeutic potential of pluripotent stem cells are inching toward realization, as several early-phase clinical studies are underway. At the same time, despite presidential declarations in favor of expanding human embryonic stem cell research, the regulatory environment is as complex as ever. Scientifically, clinically, socially, and politically, the stem cell debates are far from over. So, should federal funding for human embryonic stem cell research be expanded, diminished, or halted altogether? Ethical, legal, and regulatory issues associated with federal funding for human embryonic stem cell research in the United States have influenced the character and trajectory of the field in profound ways. These authors contend that discussions about the significance of induced pluripotent stem cells (iPSCs) for federal funding policy ought not be reduced to technical debate; moral disagreement cannot be reduced to a calculus of biological sameness--nor should it be. They also state that when scientists acquiesce to pressures to resolve problems of politics through scientific expertise, they place unsustainable burdens on science, straining its limits and compromising its ability to serve its important political role.
Descriptors: Genetics, Scientific Research, Political Issues, Human Body, Medical Research, Federal Aid, Financial Support, Ethics, Legal Responsibility, Scientists, Public Opinion, Public Policy, Democratic Values
Wiley-Blackwell. 350 Main Street, Malden, MA 02148. Tel: 800-835-6770; Tel: 781-388-8598; Fax: 781-388-8232; e-mail: email@example.com; Web site: http://www.wiley.com/WileyCDA
Publication Type: Journal Articles; Opinion Papers; Reports - Descriptive
Education Level: N/A
Audience: N/A
Language: English
Sponsor: N/A
Authoring Institution: N/A


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For content prior to 9/19/2008, use ourold search engine or browse older editions.
MADISON -- The Madison Diocesan Council of Catholic Women and Madison Catholic Woman's Club jointly invite all women of the diocese and their guests to "A Day of Christian Renewal" on Monday, Oct. 15, at St. Maria Goretti Parish Hall, 5313 Flad Ave., Madison.
This day of reflection, fellowship, and prayer begins at 9 a.m. and includes morning refreshments, program, lunch, and closes with Mass at 2:15 p.m.
Reservations by check made payable to Madison Catholic Woman's Club for $25 per person should be sent to: Day of Renewal Reservations, c/o Carol Strmiska, 4321 Green Ave., Madison, WI 53704, and received by her by October 7. Include your phone number and guest's names as well as their parish/vicariate with your reservation. The facility is barrier free.
Diocese of Madison, Catholic Herald 702 S. High Point Rd., Suite 121, Madison, WI 53719Phone: 608-821-3070 Fax: 608-709-7612 E-mail: email@example.com Web site created by Leemark Communications and Catholic Herald staff.


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To continue the discussion started in the 'Dead Pool' thread:
@ Oxy: your well-made points seem to be in response to something I did not say. (All too often liberal-minded people make a knee-jerk response whenever certain topics are aired, and fail properly to read, let alone understand, whatever is actually being said by an apparently nasty and allegedly 'illiberal' writer.)
You seem to assume that I advocate the death penalty as a deterrent, implying that I think this is a more severe punishment than life imprisonment. Far from it. It may be that the 'hang-em-&-flog-em' brigade and the 'baying mob' which many liberals like to scorn feel that way, and they want 'revenge' on murderers.
My view is different. Taking someone's life is taking someone's life, and simply locking them up until they die is taking their life from them just as surely as executing them. I feel it is just as unpleasant and every bit as uncivilised. And there are many prisoners who demonstrate every year, by their suicides, that for them even short-term imprisonment is actually worse than death.
Unfortunately some convicted criminals - a very small number - have to be kept locked up and could never safely be freed. But if you had a mad dog which bit people, would you cage it until it died, or would you have it destroyed? Which would be the more civilised and, frankly, the more honest? "Oh, I didn't kill him, he just died ... all by himself ... eventually ... so my conscience is clean, I can pretend that I did not deprive him of his life and I can walk away polishing my innocent liberal halo." A bit like walling up an errant nun!
I don't want irredeemable killers executed as 'revenge' or as some kind of greater punishment. I would be perfectly happy for them to kept alive, provided I didn't have to pay for this. And this isn't just Titus being his usual tight-arsed self; funds are limited, and I think the money involved would be much better used to improve the conditions of prisoners serving shorter sentences, in the hope of reforming a few more of them. If you want a life-sentence murderer to be fed, sheltered and guarded for decades, that's fine by me; but you pay the cost.


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Group & High Net Worth Individual Destination Travel
Brush Creek Ranch Experience | Saratoga, WY
First settled in the late 1880's as a working cattle ranch, The Lodge & Spa at Brush Creek Ranch has emerged as an iconic and supremely all-inclusive boutique resort, set up against the stunning expanse of the Medicine Bow National Forest, in the town of Saratoga, Wyoming. Our collection of historic ranchlands expands upon our proud tradition of preserving the authentic American West and sharing it with our guests for generations to come.
Southwind Plantation | Attapulgus, GA
SouthWind Has a History of Providing the Ultimate Hunting Experience
SouthWind Plantation was once a vision of founder, Tim Smith, who has transformed his vision into the "ultimate hunting experience". This vision began with an initial land purchase of one hundred seventy acres in 1994. For the first six years of operation, SouthWind Plantation was enjoyed on a private basis. In 2000, SouthWind's doors opened to the public offering the original Main Lodge for overnight accommodations.In 2005, our fifth season of public operation, SouthWind Plantation was honored to become one of only twenty-four Orvis Endorsed Wing-Shooting facilities in the United States.
Super Bowl Experience 2020 | Miami, FL
Through our partnership with the NFL, NFL On Location Experiences has direct access to the best hotels in Miami. Whether your goal is entertaining a corporate group, convenience to the game, or oceanfront views, NFL On Location Experiences has the broadest selection of the best hotels in Miami. Choose between hotel accommodations at, among others, the Biltmore, the Fontainbleau, Me Miami, the Ritz-Carlton, and the Diplomat Beach Resort.
NCAA final four 2020
April 4 & 6, 2020
Mercedes-Benz Stadium | Atlanta, GA
- Luxury Suites
- In-Stadium Club Access
- Premium Seating
- Official NCAA Experience


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Cone Man Running Productions presents A Common Martyr
Many events have been canceled or postponed due to coronavirus concerns. Please check with the organization before going to any event.
A weekend in the woods with your best buds and a cooler full of beer. Exactly what Sidney needs to help him hide when his girlfriend chases him down to get the commitment he's not ready for. Exactly what Randall needs when the girl of his dreams stops by. Exactly what Maddie needs to get brave enough to tell her crush how she's been feeling all along. Exactly what Curtis needs to tell his buddies the truth. As the empty beer cans pile up, these friends will ride the drunken roller coaster of laughter and tears, jokes and fights, kisses and crushes - all in one weekend.


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Supermarket self-service checkouts have been with us for such a long time that it's remarkable how much resistance there still is to them. I blame the supermarkets for not using the advances in technology to humanise these machines. Surely they could be enabled to identify customers and decide whether to address them as "sir" or "madam" or "love". They could also be programmed to engage in small talk, to utter a few phrases, such as "I see someone's having a stir-fry tonight" and "These biscuits look interesting" and "Nasty weather out there."
To make the experience more realistic, the checkouts could also talk to each other, exchanging such remarks as "I'm supposed to be on my break" and "They're short staffed in Fruit and Veg."
The real delight of this added sophistication will be that there is more opportunity for technical hitches and chaos in a world where we are all addressed by robot voices. As the supermarket checkout commands us to do a U-turn at the next opportunity, a motorist on the A40 will be warned of an unexpected item on the next roundabout and passengers in a lift leaving the third floor of a building in Chesterfield will hear a strange voice saying: "Those lamb chops look nice."
Imagine if train annoucements were confused with ones at the supermarket (Alamy)
As we dutifully scan our first item at Waitrose, the checkout will say: "Stand back, fast train approaching". Meanwhile, commuters waiting glumly on platform nine at Victoria station will be told that seedless raspberry jam has been cancelled and a 93 London bus will inform its restive queue: "I'm supposed to be on my break."
At some moment, with luck, the whole country will come to a standstill, with all of us poised at the checkout, motionless on the motorway, in the lift and on the train in bewildered silence. Then, throughout the land, a voice will ring out: "Please wait, the assistant is coming."


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The Best Custom Motorcycles In The World, November 20th 2014
Your weekly CliffsNotes on the best custom bikes in the world...
Redonda Racing's Suzuki TR750 Tribute to Barry Sheene
Just as notorious for his drinking, smoking, and partying as he was for winning a pair of world championships, Barry Sheene was the James Hunt of the motorcycle world, and in fact, the two were pals. This Suzuki, built by Portugal's Redonda Racing, is a tribute to the man. The build included stripping all unnecessary weight and upgrading everything from the electronics to the brakes to state of the art equipment. The best part? They actually take it to track days.
H/T: Pipeburn
Huge Design's Stoc RR "Cafe Fighter" Kit
You don't see too many custom superbikes. This one's actually a kit for the DIY type who doesn't feel like building one up from scratch. All you need is any 2009 and beyond Honda CBR1000rr—not exactly a timid bike—and boom. Equal parts cafe racer and street fighter.
H/T: BikeEXIF
Ton-Up Garage's BMW R75 Recall
It's a safe bet that any Beamer the guys at Ton-Up Garage produce is going to be lustworthy. This one's no different: everything mechanical has been rebuilt to original condition, the bike was lowered a by couple inches in front, fenders were tweaked, wheels were swapped, and by the time they were done slightly tweaking almost everything on the bike, this beauty emerged.
One Way Machine's Harley Softail "Brougham"
This is a German-built custom Harley inspired by a Cadillac. It might sound odd, but the man behind One Way Machine latched onto the Cadillac Eldorado Brougham when he first saw it, hence the name. Other than that, the stripped-down bike's the antithesis of the original Brougham, which came with everything from power windows to whisky glasses (really). You're going to want to take a closer look on this one over at BikeEXIF. It just won the world championship as the best custom Harley of 2014.
H/T: BikeEXIF
Vis Maior's Triumph Concept
Vis Maior is that Swiss collective full of bike loving graphic designers that produce first-rate custom bike visualizations so you can show your builder exactly what you want. They've worked their magic again, this time on a sweet Triumph that looks like it'd be a total blast. Someone, anyone: please build this in real life.
Aaron Miller is the Rides editor for Supercompressor, and can be found on Twitter. He's all about that Barry Sheen tribute Suzuki.


text: Swapna (Tapsee) is a recreation designer who's on a turbulent street to restoration from an incident that scarred her deeply. She finds herself within the fingers of an evil pressure that has been committing unspeakable crimes within the metropolis. On a fateful evening, Swapna has to take care of the darkish forces exterior and inside. With solely her maid (Vinodhini) for her firm how she manages the scenario is the remainder of the film. official trailer


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Voting continues till midnight in Andhra
Published on Fri, Apr 12 2019 10:51 IST | 164 Views
Kadapa: YSR Congress Party President Y.S. Jagan Mohan Reddy shows his inked finger after casting his vote for the first phase of 2019 Lok Sabha elections in Andhra Pradesh's Kadapa on April 11, 2019. Polling was underway for all 25 Lok Sabha seats and 175-member Assembly in Andhra Pradesh. (Photo: IANS)
1 / 1
RELATED NEWS: Voting continues till midnight in Andhra


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Every CalCERTS company has a Customer ID. This number can be used to search the Industry Partners lists for the company.
- Login to www.calcerts.com.
- Click on your name display located in the top right hand corner, then click My Company Info.
- The Customer ID is displayed on the first line of this page.


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Sunday, November 15, 2015
Cafe Kathmandu @ Tengkat Tong Shin
13 comments:
- AnonymousNovember 15, 2015
Tengkat Tong Shin? I always go to Muar Restaurant there, nice home-cooked kind of food...and they say the beef noodles round the corner by the roadside is very nice. Sure would look out for this one if I ever drop by again. Don't mind trying some Nepalese.ReplyDelete
I love a lot of the art there! dishes look pretty simple. Nice.ReplyDelete
Oh this is definitely going on my to visit list. Sean we're moving by the way. Please check out our new platform and tell us what you think: theyumlist.net Monica :-)ReplyDelete
Interesting and uncommon dishes... but not too sure if they work well with Red :PReplyDelete
I don't know why but I only seem to hear the song Katmandu playing in my head.....haha! :DReplyDelete
I remember I had momo at Nepalese restaurant at Sri Hartamas and I like it!ReplyDelete
Wow! Food looks awesome! Definitely gonna give them a visit soon. R the food served halal?ReplyDelete


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I haven't been on those rides, but the Bandera area is very beautiful. I sure hope this is a temporary problem. Carla Richardson Dolores, Colorado -----Original Message----- From: Michael Maul <email@example.com> To: ridecamp <email@example.com> Date: Wednesday, August 16, 2000 10:42 AM Subject: RC: CT Region Lone Star Rides canceled >The AERC office asked me to post this. Sorry to see this. I have done >these and it's nice to have a ride with hills in TX. > >Mike > >From: <email@example.com> >To: <email@example.com> >Sent: Wednesday, August 16, 2000 5:17 AM >Subject: RE: LONE STAR l, ll, lll > > >> I would appreciate your getting the word out as soon as possible that at >this >> time I will have to cancel the Lone Start l, ll, lll ride which was to be >> held the weekend of November 24-26, at Hill Country Natural Area in >Bandera >> Texas. >> >> Due to several unforseen circumstances I think it best at this time to >cancel >> due the safety and concern for horses and riders. >> >> Hopefully I will be able at a later date continue with this ride, but for >now >> it is cancelled. >> >> Thank you >> >> Debbie Allen >> > > >=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= >Ridecamp is a service of Endurance Net, http://www.endurance.net. >Information, Policy, Disclaimer: http://www.endurance.net/RideCamp >=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= > >
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Again, Metuh loses as court rejects appeal by ex-PDP spokesman
The Court of Appeal has dismissed an appeal by a former Spokesperson for the Peoples Democratic Party, PDP, Olisa Metuh, seeking to upturn the decision of a Federal High Court, Abuja, by releasing his international passport for him to travel abroad.
It would be recalled that Metuh is being prosecuted by the Economic and Financial Crimes Commission, EFCC, for alleged N400 million fraud.
EFCC spokesman, Tony Orilade in a statement said, "He had on February 22, 2017, through his counsel, Onyechi Ikpeazu, SAN, applied for the release of his passport in order to "travel to the United Kingdom for five weeks to attend to his failing health".
Earlier, Justice Okon Abang had dismissed the application as "lacking in merit", and sternly warned his counsel against deploying tactics to delay the trial, which has been ongoing since January 6, 2016.
Dissatisfied with the ruling, Metuh had approached the Appellate Court, praying for the release of his travel document.
But the Court of Appeal on January 30, ruled that his application was "an abuse of court process", as he failed to disclose cogent reasons for him to seek medical attention abroad.
Following the latest ruling, Metuh's trial before Justice Abang will continue on February 25.