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Single cell whole genome amplification is susceptible to amplification biases that impact the accuracy of single cell sequencing data. To address this, we have developed a microfluidic device for the isolation and purification of genomic DNA from individual cells. The device uses a micropillar array to physically capture single cells and its chromosomal DNA upon extraction. The extracted DNA is immobilized within the micropillar array in a way that allows isothermal amplification. In this system, whole genome amplification of the single cell is carried out under a continual fluid flow within the microfluidic channel. We have demonstrated the process for amplification of individual human cancer cell genomes from the HeLa cell line. By sampling select gene loci along the human genome and performing whole exome sequencing, we demonstrate improved genome coverage and reduced amplification bias compared to amplification of single cells deposited in wells by fluorescence activated cell sorting.
Citation: Tian HC, Benitez JJ, Craighead HG (2018) Single cell on-chip whole genome amplification via micropillar arrays for reduced amplification bias. PLoS ONE 13(2): e0191520. https://doi.org/10.1371/journal.pone.0191520
Editor: Victor M. Ugaz, Texas A&M University College Station, UNITED STATES
Received: August 29, 2017; Accepted: January 5, 2018; Published: February 12, 2018
Copyright: © 2018 Tian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Exome sequencing data has been uploaded to the NCBI SRA (Accession Number SRP127286). All other relevant data are within the paper and its Supporting Information files.
Funding: This work was funded by Cornell University.
Competing interests: The authors have declared that no competing interests exist.
Single cell analysis has become increasingly important for understanding and diagnosing disease.[1–6] For instance, cellular level aberrations have been shown to play critical roles in tumor heterogeneity, cancer metastasis, drug resistance, and cell fate.[7–12] Investigating these aberrations and differentiating between cell types within a population may give rise to improved treatments, however, single cell handling and analysis remains difficult. Because there are only picogram quantities of DNA within a single cell, existing due to sensitivity limits, existing workflows cannot sequence single cell genomes directly without amplification.[13–15] Thus, to obtain a sufficient quantity of material for sequencing, single cell whole genome amplification (WGA) is necessary. Among the most widely used single cell WGA amplification techniques is multiple displacement amplification (MDA), which relies on a combination of random sequence primers and the strand-displacement properties of the Phi29 polymerase to isothermally amplify DNA.[14,15] However, amplification bias stemming from chimera formation and non-linear enrichment remain an issue in single cell WGA through MDA.[16–18] This bias can be averaged out when analyzing monodisperse multi-cell population samples because of the multiple copies of each gene from the many cells. However, biases occurring on the single cell level lead to underrepresentation of genome regions that were not amplified early-on in the MDA reaction.[19]
To this end, several techniques have been found to minimize amplification bias during MDA by reducing reaction volumes.[20] Although the mechanism by which reducing amplification volume reduces bias remains to be fully explained, it has been demonstrated across several platforms. These platforms can be broadly categorized into limiting dilution technologies,[21] droplet microfluidic technologies,[22–24] and chambered microfluidic technologies.[19,25] Limiting dilution technologies provide a high degree of parallelism, but the microwells can suffer from cross-contamination of liquids and reagents.[21] More reliable compartmentalization of single cell genomic material can be achieved via emulsion enclosure and microfluidic chambers, however complex channel geometries and valving systems are required to achieve an integrated platform capable of both single cell isolation and genomic analysis. Hence, exploring alternative methodologies of integrating cell capture and genomic analysis is a critical component of the overall effort to improve single cell sequencing.
Recently, our group has developed a valveless microfluidic device for on-chip cell capture and DNA extraction.[26] The approach uses micropillar arrays to physically entrap genomic DNA (gDNA) from cells upon lysis. As this process is purely mechanical, it does not require any chemical modification of surfaces or cell sample preparation.
We have designed a new microfluidic structure for single cell capture and developed a process for genomic amplification via micropillar arrays (GAMA). GAMA relies on the high capture efficiency and DNA immobilization properties of the micropillar array to hold the template gDNA in a fixed position within the microchannel during processing. The genomic DNA is purified in the system because all lysed cell components, including mitochondrial DNA, are washed away while the gDNA is retained in the pillars. This approach also differs fundamentally from existing technologies in that our microfluidic chamber containing the tethered template DNA is subjected to a constant hydrodynamic flow throughout the WGA process. This constant flow also allows reagents within the channel to be continually replenished while amplified product washes downstream into the output reservoirs where it may be collected for off-chip analysis. The purification of the gDNA achieved during the preceding gDNA extraction step also reduces artifacts in the resulting WGA product pool. To characterize our approach, we compare genome coverage through gene loci sampling and whole exome sequencing (WES) between GAMA and FACS based methods.
Cell culture
HeLa-GFP cells (Paul Soloway Lab; Cornell University) were cultured in Dulbecco's Modified Eagle medium (DMEM) (Invitrogen) within a T75 flask at 37C and 5% CO2. Cell culture medium was supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals; Atlanta, GA), 1% (wt) non-essential amino acids (NEAA) (Gibco, Life Tecnologies), 1% (wt) L-glutamine (Gibco, Life Tecnologies), 2% (wt) HEPES (Quality Biological; Gaithersburg, MD), and 0.001% 2-mercaptoethanol (βME) (Sigma-Aldrich; St. Louis, MO). Cells were passaged at 60% (vl) confluency roughly twice per week.
Device fabrication
Silicon molds for polydimethylsiloxane (PDMS) microfluidic devices were fabricated using standard photolithography techniques. Briefly, wafers (Ultrasil; Hayward, CA) were spin coated with Microposit S1813 photoresist (Shipley; Marlborough, MA). Device pattern was transferred onto photoresist layer using UV contact lithography (ABM contact aligner, ABM-USA; San Jose, CA). Exposed substrates were developed in 726MIF developer (Microchemicals). Microfluidic pattern was transferred onto top silicon layer by Bosch process in a Unaxis SLR 770 deep reactive ion etching system (Unaxis USA Inc.; St. Petersburg, FL). Etch depth was determined to be 20–25 μm using a P10 profilometer (KLA Tencor; Milipitas, CA) and a Zygo otical profilometer (Zygo Corporation; Middlefield, CT). A monolayer of (1H,1H,2H,2H-Perfluorooctyl) Trichlorosilane was deposited on the etched wafers in a MVD100 molecular wafer deposition system (Applied Microstructures; San Jose, CA) to prevent adhesion of PDMS to the mold. Sylgard 184 (Dow Corning; Midland, MI) PDMS base resin was mixed with the curing agent at a 10:1 ratio, degassed under vacuum at room temperature, poured onto the master, and cured for 45 minutes at 150°C. The elastomer casting was then peeled off the mold and access holes to the input and outputs of the microchannels were created with a 1.5 mm biopsy punch (Sklar Instruments; West Chester, PA). To complete channel fabrication, the patterned PDMS was treated with air plasma for 1 minute and bonded to a 500 μm thick fused silica wafer (Mark Optics; Santa Ana, CA). Experiments were run with the microfluidic device mounted on the stage of an Olympus IX-70 inverted microscope (Olympus; Center Valley, PA) to image and observe the microfluidic channels in real time.
Cell capture and lysis
HeLa-GFP cells were trypsinized from T75 flasks with 0.25% Trypsin. Trypsinized cells neutralized with 1:1 dilution of media, spun down in a centrifuge, and then resuspended in fresh PBS at a concentration of 1:50. The cell suspension was flowed into the microfluidic device via pressure driven flow at 0.5 psi with bone-dry nitrogen gas (Airgas; Radnor Township, PA). The infusion apparatus was then disconnected from the microfluidic device's input port, washed with alternating cycles of ultrapure water (Invitrogen; Carlsbad, CA) and 100% ethanol to remove the remaining cells within the reservoir, and then reconnected to the microfluidic device input port. Sterile PBS buffer was then flowed into the microfluidic device for 5 minutes to allow uncaptured cells to either be arrested within the cell capture region or to flow through the device into the output reservoirs. The output reservoirs of the device were then emptied and rinsed with ultrapure water. The entire device is visually inspected for any potential cells lodged outside of the micropillar array. Channels that have trapped multiple cells are discarded in post-amplification analysis. Similarly, in cases where a cell has become lodged upstream to the channel divider, the entire device is discarded, and the experiment is ended to prevent inaccurate analysis resulting from contamination. Lysis buffer comprised of 6M guanidinium thiocyanate (Sigma-Aldrich; St. Louis, MO) in water was flowed into the microfluidic device for 5 minutes also by pressure driven flow at 0.5 psi. After visually confirming cell lysis in all ten channels, the lysis buffer was removed from the input reservoir and the entire microfluidic device is flushed with 100% ethanol for 5 minutes. The ethanol is replaced by washing with ultrapure water for 5 minutes and then finally replaced by PBS buffer. The output reservoirs that now contain a mixture of cell lysates, lysis buffer, ethanol, water, and PBS was then emptied and cleaned via rinsing first with 100% ethanol and then ultrapure water. The genomic DNA tethered within the microfluidic device is now ready for whole genome amplification.
On-chip whole genome amplification
Whole genome amplification (WGA) of the single cell genomic DNA tethered within the micropillar array region of the microfluidic device was carried out using reagents from the REPLI-g UltraFast Mini Kit (Qiagen; Hilden, Germany). Prior to starting the reaction, 280μl of buffer D1 was made by adding 35μl of buffer DLB to 245μl of ultrapure H2O. 400μl of buffer N1 was then prepared by adding 40μl of stop solution to 360μl of ultrapure H2O. Finally, 288μl of master mix was made by adding 18μl of polymerase to 270μl Repli-G UltraFast reaction buffer. To denature the double stranded gDNA tethered on the micropillar array, buffer D1 was flowed through the device continuously at room temperature for 8 minutes. Buffer D1 was then removed and the device was flushed with buffer N1 for 15 minutes. Afterwards, both the infusion apparatus and the ten output reservoirs were emptied and washed with 100% ethanol and then ultrapure water. The infusion apparatus was then loaded with the master mix solution and pressure was set to 0.5 psi. Pressure was then held constant throughout the entire duration of the 3.5 hour reaction amplification reaction while the device was placed atop a hot-plate set to 33C. After the reaction was completed, 5μl of ultrapure water was added to the amplified DNA product at each output reservoir. The amplified genomic DNA in the reservoir was then pipette collected from each output reservoir off-chip into a polymerase chain reaction (PCR) tube. All samples were heat inactivated at 65C for 10 minutes and placed in a -20C freezer until further use. Sample yield was measured using Qubit 2.0 Fluorometer (ThermoFisher; Waltham, MA) with dsDNA HS dye kit at a 1:200 sample dilution.
FACS single cell WGA
A FACS machine (Becton Dickinson Biosciences; San Jose, CA) was used to sort single HeLa-GFP cells into a PCR-compatible microwell plate (Bio-Rad; Hercules, CA) with each well containing 5μl of sterile PBS buffer. The microwell plate was then spun down in centrifuge at 1000G for 5 minutes to ensure that sorted single cells were sitting at the bottom of their respective wells. Buffer D2 (buffer D1 supplemented with 10% Dithiothreitol for cell permeabilization) and master mix were then prepared according to the Repli-g UltraFast kit's protocol. To lyse the single cells in each microwell, 5μl of buffer D2 was added to each well and incubated on ice for 10 minutes. 5μl of stop solution was then added to each well and incubated on ice for 5 minutes. Finally 53.3μl of master mix was added to each well and the microwell plate was placed in a thermocycler (Eppendorf; Germany) set to hold at 30C for 3.5 hours.
Gene loci PCR
Primers were designed to target 150bp-200bp regions within six gene loci (ERBB2 17q12, PRMT2 21q22, P53 17p13, CCND1 11q13, TRAM1 8q13, and MyC 8q24) and ordered through Integrated DNA Technologies (IDT; Coralville, IA). Lyophilized primers were dissolved in water to a concentration of 10μM. Then, following the protocols from the Taq DNA Polymerase Kit (Life Technologies; Carlsbad, CA), 50μl reaction were prepared for each of the 6 gene loci for every collected single cell WGA sample. 30 cycles of PCR were carried out and the PCR product was run on a 2.3% agarose gel via electrophoresis. Using a 2-log ladder (New England Biosciences; Ipswich, MA), the appropriate size region of 100bp-200bp was evaluated for the presence or absence of the gene.
Whole exome sequencing
Whole exome sequencing (WES) was performed via the Weill Cornell Medical College sequencing facility. Samples libraries were prepared using Agilent SureSelect Target Enrichment Kit (Agilent; Santa Clara, CA). Exome pulldown was performed using SeqCap EZ Exome v3 Human Exome Kit (Roche Sequencing; Pleasanton, CA). Samples were sequenced in a single lane on a HiSeq 4000 (Illumina; San Diego, CA) with paired end clustering 100x2 cycles. Resulting reads were aligned to human reference genome HG19.
Results and discussion
Channel design and experimental setup
Fig 1(A) shows the overall experimental setup for GAMA. To create the chip device, a slab of mold-casted PDMS (polydimethylsiloxane) imprinted with the channel geometry is bonded to a glass slide to create the microfluidic device. Reagents are loaded into the device via pressure driven flow from an infusion apparatus housing a large fluid reservoir. Fluid that is loaded into the infusion reservoir can be easily exchanged and replaced via pipetting. The infusion apparatus is a two-part mechanism consisting of a reservoir portion that can be connected to the microfluidic device via an input port and a cap that is connected to a nitrogen source used to drive channel flow. Fig 1(B) shows a top down view of the device design. The GAMA device has a single input port and 10 separate output ports allowing multiple single cell samples to be run in parallel. These ten channels each contain identical designs consisting of a single cell capture region and micropillar array Fig 1(C). HeLa cells expressing green fluorescent protein (GFP) were selected as a model cell type for our experiments due to the ease of handling and fluorescent properties. To show the device in scale, Fig 1(D) shows four such devices can be casted from a 4-inch silicon wafer mold as a single slab and bonded to a glass-silica wafer.
(A) Exploded view of our PDMS microfluidic device. An infusion apparatus is connected to the single input port to provide pressure driven flow of fluids and reagents. (B) Device schematic showing valveless 10-channel device design in which product from each channel can be collected separately. (C) A micrograph of the single cell capture region and micropillar array. (D) Picture taken of 4 separate 10-channel devices made from a single PDMS slab bonded to a 4-inch diameter glass silica wafer. (E) Micrograph showing single cell capture, and (F) subsequent lysed cell imaged under fluorescence with YOYO-1 intercalating dye staining of genomic DNA immobilized within the pillar array region.
Single cell GAMA
As depicted by the graphic in Fig 1(E), the single cell capture region consists of a series of 1.5μm posts spaced 2μm apart and arranged in an orientation to arrest cells in the apex of the micropillar array. The size and spacing of these posts were selected to be small enough so that the genomic chromosomal DNA from the trapped cell would become entangled within the array due to their length while smaller molecules such as RNA, proteins, and lipids would flow through the array unhindered. The height of the microchannel is adjusted to the size of the cells to prevent multiple cells from stacking on top of one another in the Z-plane. Furthermore, barriers enclosing the micropillar array also aid in preventing cells from lodging in the array downstream of the desired capture region. Occasionally, multiple cells are captured within the micropillar array or become adhered to the glass surface within the microchannel. To reduce cell adhesion on the channel surfaces, we increased flow rate during cell loading to be above 2μl min-1. This flow rate was then reduced once the cells were loaded and prior to cell lysis to 0.5μl min-1. Channels containing multiple cells are discarded in the analysis and the entire device is visually inspected prior to cell lysis to ensure that there are no cells lodged outside of the desired cell capture region.
Upon introduction of lysis buffer, the micropillar array will physically immobilize the gDNA. This immobilization process occurs as a result of the chromosomal DNA being physically entangled on the pillars due to their centimeter scale lengths, while smaller cellular components such as lipids, proteins, RNA, and mDNA are washed away downstream. The immobilized gDNA can be imaged via fluorescent staining with DNA intercalating dye labels such as with YOYO-1 shown in Fig 1(F).
After cell capture and lysis, the extracted gDNA tethered within the micropillar array was amplified via MDA under a continuous flow of 0.5μl min-1. An illustration of the GAMA workflow can be seen in Fig 2. Although we had initial concerns that denatured DNA strands would rapidly reanneal prior to amplification, we found GAMA to yield close to a 10,000-fold amplification as measured by final DNA concentration of the amplified product. Another concern was that the highly branched structures characteristic of isothermal amplification with Phi29 would become entangled within the pillar array and occlude the flow, however we did not observe any product buildup or clogging upon inspection with DNA fluorescence imaging post-amplification. We reason that this is because the average sized fragments produced from the MDA, roughly 12kb in length, are too short to tether around the 1.5μm diameter micropillars without slipping off.
Here, a view of the inside of the sealed microfluidic platform is shown. (A) Fluid flow (blue arrow) moves from left to right in this schematic and fluids can be exchanged within the input reservoir to control the local environment within the channels. (B) Artist depiction of a single cell that is trapped within the apex of the micropillar array. Side walls enclosing the micropillar array guide the cell towards the apex of the array while excess cells bypass the pillar array to the output reservoir. (C) Upon cell lysis, genomic DNA (orange) from the trapped cell will become entangled in downstream pillars while debris from the cell lysate is washed away. Reagents for whole genome amplification is flowed into the channel and as amplification occurs, product DNA strands (blue strands) are carried downstream and collected in output reservoirs.
Validation and gene loci detection
Because the MDA kit we used has been shown to produce artifact DNA, simply quantifying the amount DNA collected from the output reservoirs is insufficient to determine the success or failure of on-chip single cell GAMA. Hence, to validate the GAMA technique, 6 different cancer gene loci along the human genome were selected as sampling intervals to ensure successful amplification of that genome region. Using the product collected from GAMA as a template for PCR, the presence or absence of each of the 6 gene loci was evaluated as an initial means of assessing GAMA performance.
Fig 3 shows micrographs taken from the cell capture region of the 10 channels in a single device. Channels containing a single HeLa-GFP cell (2, 5, and 7) were compared to channels with 0 cells (1, 3, and 10) in the number of gene loci detected post GAMA. Samples from channels such as channels 4, 6, 8, and 9 are disregarded due to having multiple cells. The capture of multiple cells in certain channels are due to unoptimized channel dimensions and micropillar spacing for the cell type used and occasional occurrences of cell adhesion onto the glass surface due to non-specific binding. In future iterations of the device, non-specific cell adherence can be mitigated through treating the glass surface with blocking agents such as bovine serum albumin (BSA) protein buffers or charge-shielding the channel with chemicals such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). While it was found that many of the gene loci were present in the amplification product collected from single cell channels, as shown by Fig 4, no gene loci were detected in empty channels on the same device. Thus, this indicates that there is no cross-channel contamination or upstream contamination contributing towards our results.
Cell counts for each channel are labeled in yellow text. The device is visually inspected for unaccounted cells lodged outside of the pillar array and channels containing multiple cells are discarded. Channels containing single cells (2, 5, &7) are analyzed in comparison to empty channels (1, 3, &10), which serve as negative controls from the same device to further assure the WGA product from each channel does not contain any potential upstream DNA contamination.
Using GAMA, single cell WGA covers up to 6/6 targeted gene loci versus 2/6 when using conventional single cell WGA with cell isolated via FACS. In-channel single cell negative controls with no cells expectedly show no gene loci detected, and off-chip bulk-level analysis affirms the specificity of primers used in loci detection.
Comparing GAMA to FACS
In order to assess amplification bias and overall genome coverage we compared gene loci detection between single cells amplified by GAMA to single cells amplified with conventional assays. For the conventional assay route, we used Fluorescence Activated Cell Sorting (FACS) to isolate single cells into PCR-compatible 96-well plates and amplified the gDNA from these cells using identical kits, reaction times, and reagent batches as those used in GAMA experiments. Our findings, shown in Fig 4, is that when using the same reagents and WGA parameters as single cells amplified by GAMA, only one to two gene loci were successfully amplified from the FACS isolated single cells versus 4 to 6 loci successfully amplified by GAMA.
Further expanding upon the gene loci detection results, we submitted single cell whole genome amplified samples from both GAMA and FACS for WES. In sequencing, we found that single cells amplified by GAMA had a higher percentage of mapped reads compared to single cells isolated through FACS and amplified by conventional assays (highest percentage of mapped reads 98.5% for GAMA versus 65% for FACS). Additionally, as shown in Fig 5, we found that although FACS samples had a higher frequency of low depth (≤3) reads compared to GAMA samples, FACS samples experienced a sharp drop off in read count as depth increased. In contrast, GAMA samples exhibited a much more gradual decline in read count as a function of depth. This suggests that the GAMA process amplifies single cell genomes with higher uniformity than conventional WGA assays. However, as we maintained identical reagents and reaction times between FACS samples and GAMA samples, we attribute the differences in amplification uniformity to a few key differences between GAMA and conventional WGA assays.
The two FACS isolated single cell samples whole genome amplified by MDA exhibit more of low depth reads with a sharp drop off in read count before reaching 10x coverage. In contrast, GAMA single cell samples showed no significant drop in frequency of reads as depth increased and showed more uniformity across the genome.
The first of these advantages is in performing amplification under a constant flow. Doing so allows amplification reagents to replenish the local template environment more rapidly than diffusion dependent systems. Flow higher than this add no reaction advantage, but sufficiently high flows could shear or dislodge the DNA from the pillars. Our selectin of flow rate in this stage of the process included considerations of minimizing the use of costly reagents. Moreover, another advantage conferred by performing amplification under a constant flow is that the amplified product DNA is carried downstream by fluid flow away from the immobilized template once the polymerase detaches from the template, thereby possibly reducing or prevent chimera formation. Secondly, as the template gDNA is linearized throughout the amplification process in GAMA, polymerase molecules have increased overall accessibility to the template whereas the gDNA in conventional assays would be in a coiled conformation thereby biasing towards binding of polymerase on the gDNA away from the center of the coil. Finally, the third advantage of performing single cell WGA with GAMA lies in the micropillar based cell lysis and DNA extraction process. As the GAMA cell lysis process uses size-based selection to physically capture gDNA within the micropillar array while smaller molecules such as lipids, proteins, RNA, and mDNA are filtered out of our device, GAMA essentially provides a purification of the gDNA template prior to WGA. In our comparisons, we found FACS samples to contain over two orders of magnitude more mDNA than GAMA samples (>20,000 mDNA reads for FACS; < 150 mDNA reads for GAMA).
Although whole genome sequencing will be required to further characterize single cell GAMA, the overall coverage breadth of GAMA for all targeted exomes (81.08% at ≥1x depth; 70.35% at ≥10x depth) was comparable to existing single cell WGA methods such as Multiple Annealing and Looping Based Amplification Cycles (MALBAC) and Emulsion Whole Genome Amplification (eWGA).[23] However, as we did not carry out independent WES trials using MALBAC or eWGA, we refrain from venturing conjectures towards the source of differences between these methods and GAMA.
We have described a simple, valveless, and scalable micropillar-based microfluidic device capable of on-chip single cell processing and WGA. Unlike conventional single cell platforms, the GAMA platform performs WGA under constant flow on purified and immobilized gDNA. Although we have yet to optimize these flow rates and their effects on the final amplification performance, we have taken the first step to validate the viability of the GAMA approach by demonstrating reproducible on-chip amplification without upstream or cross-channel contamination. Through gene loci PCR, we report that GAMA reliably amplifies more regions of the genome from single HeLa cells as compared to conventional assays with single cells isolated through FACS. Finally, we determined by WES that GAMA has improved amplification uniformity across the genome and greatly reduced levels of non-specific amplification, such as amplification of mDNA. Together, these findings suggest GAMA may be used as a platform to overcome current bias-based limitations experienced by conventional single cell assays and improve the accuracy of single cell analysis.
Supporting information
S1 File. Primer sequences used in gene loci detection.
S2 File. Gene loci detection gel images.
We thank Dr. Peter Schweitzer of the Cornell Biotechnology Resource Center Genomics Facility for initial help with DNA sequencing. We also acknowledge Dr. Sharon Soueid-Baumgarten and Professor Ben Cosgrove for their help with single cell isolation through FACS as well as the lab of Professor Paul Soloway for providing the HeLa cell lines. Microfluidic devices were fabricated in the Cornell Nanoscale Facility (CNF) and FACS was performed in the Cornell Weill Hall Imaging Facility as a part of the Cornell Institute of Biotechnology. Exome sequencing was performed in Weill Medical College.
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text:
Tulloch is to construct a new jetty at Holmsgarth North in the Shetland Islands for Lerwick Port Authority.
The L-shaped jetty will cost £16.5M to build and will provide deeper berthing and more working area for the fishing fleet, reports the Shetland Times.
It will extend to 800m, with 10m of water depth alongside.
The outer arm of the L-shape will create a dock sheltering a planned new whitefish market at Mair's Yard. It will also be suitable for berthing offshore industry vessels.
Tulloch will begin site set-up at Holmsgarth North before Christmas and the main works will start in the new year, with completion scheduled for autumn 2016.
The jetty was designed by consultant engineer Arch Henderson.
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Phone: 423-737-5070


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Arthritis is a common condition that causes pain and inflammation to the joints and the tissue surrounding the joints. Arthritis is characterized by swelling, pain, stiffness, and decreased range of motion. Arthritis affects over 50 million adults in the United States.
Most cases of arthritis can be caused by wear and tear, natural aging, or may develop after an injury. However, lupus arthritis and rheumatoid arthritis are autoimmune disorders.
The most common type of arthritis is osteoarthritis, the result of wear and tear through aging. Age is not always the factor, as overuse can also cause arthritis. For example, professional athletes may develop arthritis-related symptoms earlier than someone who works a sedentary job.


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A week of wood turning for experienced woodturners
Nothing to your head. Only pieces of wood where something beautiful must be made from. A week of wood turning in a prime location. Coffee, tea and lunch are included. That's delicious!
What are we going to do?
You can make whatever you want. Anyone who is involved can make something different. You decide what you make and you can ask questions or ask for advice at any time. All tools are here and can be used. Whether you want to make a series of pepper mills or the lighthouse of Ameland on a scale… Everything is possible. If your project needs special wood and you can't get it yourself, please let me know and I will try to find it.
Where and when?
This workshop is held from May 13 to May 17 from 9.00 to 17.30 hours. Coffee/tea/lunch, the wood and the use of the tools are of course included in the price. A maximum of 6 people can participate. Tip for staying overnight: At the end of the street is a campsite "Het Jagershuis". You can walk to the workshop every day or come on your bike…. http://www.campinghetjagershuis.nl/
For more information: Contact or klik hier voor meer informatie.


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EXCLUSIVE: I'm sick, not drunk... Sacked Fulham coach Wilkins reveals he is suffering with the same illness as United's Fletcher
- Wilkins reveals he is fighting the chronic bowel disease ulcerative colitis
- Former midfielder keen to dismiss rumours he has a drinking problem
- Kevin Pietersen invited to play for MCC at Lord's against Rest of the World
- Corporate hospitality packages for Rugby World Cup selling very well
Ray Wilkins, sacked as assistant manager by Fulham this week, has revealed he is suffering from the same debilitating illness as Manchester United midfielder Darren Fletcher.
Former Manchester United and Chelsea star Wilkins, 57, has been fighting the chronic bowel disease ulcerative colitis since 1990 along with periodic bouts of depression.
Wilkins has decided to reveal his medical condition in order to save his career in football management and rebut drinking allegations.
Revealing all: Ray Wilkins revealed he is fighting the chronic bowel disease ulcerative colitis
Claims were made from inside Fulham that Wilkins was the worse for wear in the dug-out for the 3-2 home defeat by Liverpool last Wednesday — a result that led to manager Rene Meulensteen and his back-room staff's dismissal.
Wilkins explained that his medical condition can give him the 'woozy' appearance of being drunk.
'I need to control the ulcerative colitis with strict medication, which I had not done before the Liverpool game,' he said. 'That is the reason I didn't return to the dug-out after half-time as I needed to be close to a toilet.
Keeping quiet: Wilkins said he did not tell Rene Meulensteen about his illness that make him appear 'woozy'
I can look the worse for wear but it is nothing to do with drink. I did
have verbals with Brendan Rodgers, but it was nothing.
'I was face to face with (Fulham) chief executive Alistair Mackintosh and the owner Mr Khan straight before the game. And if I had been unable to carry out my duties, they had the right to sack me on the spot. I most certainly hadn't been drinking on the train before the match.'
A clearly agitated Wilkins was involved in a touchline row with Liverpool boss Rodgers at half-time.
Fulham sources suggested that Wilkins had arrived at Craven Cottage not
in the best condition having earlier travelled to and from Birmingham
by train to watch Fulham Under 21s play Aston Villa.
Watching on: Fulham owner and chairman Shahid Khan (centre) and chief executive Alistair Mackintosh made changes to the managerial team last week
Tough time: Manchester United midfielder Darren Fletcher also suffers from ulcerative colitis
More from Charles Sale for the Daily Mail...
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was banned from driving for three years after a drink-driving
conviction when he was found to be three times over the legal limit.
Fulham insiders also said that Meulensteen had tolerated Wilkins not being in the best shape for work on other occasions - seen as one of the reasons for the Dutchman being sacked after just 13 Premier League games.
The club's sensitivity about the conduct of their management that night was due to American owner Shahid Khan being in attendance. And some of the players had voiced concern about whether Wilkins was well enough to carry on coaching.
Wilkins added: 'I didn't want to make a fuss about my illness, I didn't tell Rene. Darren Fletcher said how hard it was to tell his team-mates. But I desperately want to carry on in football and there's no chance of that happening if everyone gets the wrong impression that I have a drink problem.
'I was very stupid
about the drink-driving and I have had trouble with depression on
occasions. But not since I was back in football. I was so excited about
the Fulham challenge, but it's all about results.'
Also leaving Fulham along with head coach Meulensteen, technical director Alan Curbishley and Wilkins are first-team coaches Jonathan Hill and Mick Priest, who were brought from Manchester United by Meulensteen.
Will Perry, the BBC's Olympic Games reporter, should really look at his own puerile Twitter postings before using social media to criticise proper journalists in Sochi.
Getting it wrong: BBC's Will Perry made some remarks on Twitter which he later delete
Perry, an Old Harrovian heir to a family fortune, thought it appropriate to make banal fun of German ski jumper Andreas Wank's name. The BBC made no comment but the relevant Twitter comments were deleted immediately they were made aware of them.
Lord's have officially invited England outcast Kevin Pietersen to play in their showcase MCC v Rest of the World match on July 5 to mark 200 years of cricket on the ground.
Pietersen has yet to reply, but the MCC's decision to pick him so soon after he was dumped by the national selectors is not likely to go down well with the England Cricket Board, also headquartered at Lord's.
Back at the home of cricket? Kevin Pietersen has been invited to play for the MCC against the Rest of the World at Lord's
Especially as Rest of the World captain Shane Warne can be guaranteed to use the platform to berate England yet again for ditching KP and keeping Alastair Cook as captain.
The 2015 Rugby World Cup has had a difficult time organising their venues, especially with Old Trafford opting out at the last moment. But the corporate hospitality and international travel packages have gone off the scale in the first six weeks on the market.
The £30million of business is more than the London Olympics or the Brazil World Cup managed in a similar period.
Selling well: Corporate hospitality packages for the 2015 World Cup have been selling well
Meanwhile, Events International being
fined £12,000 for the illegal selling of tickets at London 2012 has not
prevented the company being appointed the official corporate
hospitality operators for the Comonwealth Games.
A Glasgow 2014 spokeswoman said: 'The unauthorised ticket selling was not in any way deliberate and compliance procedures were fully reviewed to ensure such an occurrence would not arise in future.'
Most watched Sport videos
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- Solskjaer: 'We were confident we found the right team selection'
- 'We need more determination': Maurizio Sarri after loss to Man Ut
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- Arsene Wenger raises questions over Mesut Ozil effort level
- Fabio Capello says Wayne Rooney didn't play well at Everton
- Lindsay Vonn calls Laureus World Sport Awards ovation 'overwhelming'


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You don't have to browse Instagram long before you see images of people in dangerous places. You can satisfy the thrill-seeking selfie shooter with PhotoPie's vertigo-inducing Bird's Eye View floor mat. The bottom is made of non-slip rubber that will stay in place once it's on the floor. The top material is felt and is imaged using a fade-resistant dye-sublimation process. Sizes range from 4 x 5 to 8 x 10 feet.
I was really excited to get this backdrop for my new YouTube channel (Nia Knows Best) and it did not fail. The quality is thick (which is good), and it is NOT transparent. That's perfect because I have a wall of magazine pictures behind this backdrop and yet you can't see anything but the shimmer of the rose gold glitter. The only thing I would say is that depending on the lighting the rose gold turns into a silver ish glitter which can be a little frustrating. In the last photo you can see how just because of the lighting switching, the rose gold turns into silver. Other than that it is perfect.
6× Photo Studio Light Photography Background Clips Backdrop Clamps Peg Package Contents: 6 clamps Description: These Set of 6 Universal Pro Clamps are easy to use and would be a great addition to your studio. These clamps are extremely useful in positioning or stretching your background onto backdrop support systems. Clamps are perfect when you need to attach any kind of fabric background to a support system. They work perfectly for Muslin, paper, and even canvas backdrops. These clamps are also known as spring clamps, A-clamps, and even studio tongs.
Great for the price point! Will get lots of use out of it. Biggest issue was setting it up, theres absolutely no instructions that comes with this set. There is a piece of paper titled "instructions" BUT it's literally just a list of what's inside the packaging and not how to put it together. Had to watch a video online to figure it out. This was my first time setting up something like this. Also the polls are a little flimsy and I'm always worried about it falling over however again you pay what you get, don't expect top of the line equipment when you're purchasing relatively cheap stuff. Anyways thumbs up from me, it's basically exactly what I needed.
I've included what the backdrop look like without people in it, and also what it looks like with several different kinds of skin tones so those of you who are thinking about buying this can take that into consideration with your potential clientele. As you can see, at least with just a horseshoe flash, people with darker skin tones definitely looked better with this backdrop.
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Grab a prop and get ready to strike a pose! For every reason and every season, we offer a variety of photo booth backdrops and photo booth supplies. If you're hosting a party, you know how important it is to keep guests engaged with fun activities - it's the key to taking any celebration from drab to fab! And the best way to keep the good times rolling is by setting up your very own DIY photo booth. For birthdays, weddings, holiday celebrations, graduation parties and so much more, you're bound to find the right fit for your shindig with our huge assortment of photo booth props, accessories and backdrops. Whether you're the kind of guy or gal who like to grin cheek-to-cheek or maybe you'd rather throw up a peace sign or pose with a prop, this collection of party supplies helps everybody get camera-ready. Selfie lovers, rejoice!
Display your photographic backdrops, floors, and floordrops using durable support equipment and accessories. You'll find a variety of stands and support kits that come with everything you need for mobile photography projects and studio setups. Many styles feature telescoping crossbars and adjustable bases to suit a variety of image heights and widths. Some support systems also feature multicolored chains so you can easily select the right background for your needs.


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Bora Bora ruffled cotton-blend poplin blouse 1043913 WVYSRQG
Product Code: WVYSRQG
Availability: In Stock
Availability: In Stock
$42.48 $59.88
class="editors-notes js-accordion-tab accordion-tab" open name="Editor's Notes">EDITORS' NOTESJohanna Ortiz always knows how to add sophisticated volume to a piece without making it feel over the top. Trimmed with a row of tasseled ties along the front, this 'Bora Bora' blouse is made from cotton-blend poplin that's crisp enough to hold the fluttering ruffles and has a shirred hemline that balances the silhouette. Pair yours with the label's striped maxi skirt.Shown here with: Cult Gaia Clutch, Ancient Greek Sandals Sandals, Laura Lombardi Earrings.- White cotton-blend poplin- Ties fastenings through front - 97% cotton, 3% elastane- Dry clean
Bora Bora ruffled cotton-blend poplin blouse 1043913 WVYSRQG


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All online calculators
- Kinematics. Throwing body up problem
Kinematics problem from the Svetozarov's book for MEPI matriculates
- Labor retirement pension amount (for Russia)
Calculates labor retirement pension amount in accordance with the law.
- Lagged scatterplot
This online calculator builds scatterplot of the time series against itself offset in time
- Laminate flooring. The calculation of the number of planks
The calculation of the number of planks requiered
- Land tax
Calculates the land tax
- Large breed puppy weight gain recommendations
Large breed puppy weight gain recommendations
- Least Common Denominator for several fractions
Least Common Denominator for several fractions
- Length of service calculation
Length of service calculation for several periods of work
- Lengths of triangle sides given one side and two angles
This online calculator determines lengths of triangle sides given one side and two angles
- Level of liquid in the cylindrical tank, AKA Quarter-Tank Problem
This online calculator finds the height above the bottom of a horizontal cylinder (such as a cylindrical gas tank) to which the it must be filled for it to be full at specified percentage (for example, one quarter full amounts)
- Levenshtein Distance
Online calculator for measuring Levenshtein distance between two words
- Linear Diophantine equations
This calculator solves linear diophantine equations.
- Lines filter by number
This online calculator filters lines of text by removing designated lines or, you can say, leaving only designated lines
- Liquid level in the tilted cylinder
Сalculates the level of the liquid in a tilted cylindrical tank with the tank parameters and the liquid volume.
- List filtering
Calculator that allows you to filter the initial list with the specified multiple filters
- List format
Format a list using regular expressions
- List intersection
This calculator parses list using regular expression, then counts unique matches and lists them in decreasing order
- List Join
This online calculator can join two csv lists by key column the same way JOIN works in SQL (inner join, left outer join, right outer join)
- List reverser
This online calculator reverses lines of text
- LL1 grammar analysis. Yet another top-down parser generator.
The calculator checks LL1 grammar correctness, parses a text using the grammar, shows FIRST, FOLLOW and FIRST PLUS sets, parsing tree and gives PLANETCALC parsing code.


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EXCLUSIVE: Is Romney removing Medicare Taxes for Small Business?
An unconfirmed source deep within the Romney camp has hinted at an explosive announcement next week that will be of great interest to small business owners across the country. Another unconfirmed source exclusively told BusinessBlogsHub.com that the announcement was related to the possible reduction of medicare taxes.
An unconfirmed source deep within the Romney camp has hinted at an explosive announcement next week that will be of great interest to small business owners across the country. Another unconfirmed source exclusively told BusinessBlogsHub.com that the announcement was related to the possible reduction of medicare taxes.
Medicare tax & small business no more?
Its about time I come clean and state that the above is purely fabricated by me – but it got your attention right?
I could have kept going on and provided even more 'unconfirmed' rumours about this possible 'exclusive' and researched a bit more to provide even more facts that Romney could reduce taxes, one day, or maybe not.
Then with a bit of luck major blogs like Gawker and HuffPost believe my post (or not) and create their own 'exclusive' citing this blog as the source. Then the more talk generated by the press on the fabricated 'exclusive' I created could end up as a 'confirmed' statement mentioned on TV – then its fact.
I thought this type of media manipulation was impossible to happen. Yes smaller blogs could write all sorts of BS but the major blogs like HuffPost and Gawker would take one look at it and laugh. They have fact checkers and editors screening the content before it gets published right?
But it seems this is not the case. 'Facts' can be created out of thin-air by anyone and end up as fact. Ryan Holiday can prove this works because he has manipulated the media so many times for his clients that he wrote a book about it – 'Trust Me, I'm Lying: Confessions of a Media Manipulator'.
I always thought there was something dodgy about mainstream media and their 'candy-floss' content which is mostly made up of press releases massaged to look like 'news' but Ryan exposes the true depth of manipulation in his book. Without giving up too much Ryan tells us how the system works:
- You want some press for yourself or your client so you create an idea or theme that, if believed, will generate exposure for free.
- You feed this idea/theme to a low-level blog that is desperate for any story idea. To help the blogger along you make sure that the idea when published will bring in traffic because traffic is the main currency of blogs. Traffic equates to ad impressions which equals money.
- Once the low-level blog has published the post other blogs higher up the food chain use the post to create their own 'exclusive' and possibly pad it with some facts that 'kinda' enforce the truth behind the idea or maybe out of pure luck there is another statement that re-inforces my own idea.
- From there the idea spreads to other blogs and by the time you are up to your third coffee of the day its on Gawker and HuffPost and once its on these types of sites its mostly considered fact by the general public.
The above steps are missing out a heap of information that Ryan's book provides. Ryan also provides many examples of how he used the system to manipulate the media and I have been able to search for the articles online that he seeded with his strategy.
If you are a blogger, PR person or deal with the media in anyway then read his book. Its an eye opener to how the world of 'news' really works.
Trust Me, I'm Lying: Confessions of a Media Manipulator
NOTE: this is not an affiliate link and I receive no benefits from Ryan or anyone for providing this post.
Small Biz, Big Name: The 5 Most Effective Marketing Strategies for Small Businesses in 2019
Running a local business is tough. So tough, in fact, that 50% of small businesses fail within five years of startup.
With such a competitive marketplace and with so many challenges, it has never been a better time to think about your marketing strategy.
Having effective marketing strategies will help your business stand out from the crowd and set you ahead of your competitors.
However, marketing techniques change all the time, and it's possible there are new techniques that could help your business reach out to even more customers. So, check out these effective marketing strategies to make sure your business stays ahead in 2019.
1. Facebook Ads
If your small business does not advertise on Facebook, you should reconsider. In fact, two million businesses advertise on Facebook, so there must be something in it!
Facebook ads are an inexpensive way to reach a wide audience. They also have the added bonus of targeting to your chosen audience demographics and location.
Creating a Facebook ad is also pretty quick and easy and does not require specific skills. All you have to do is create a regular Facebook post with images or video to upload, and away you go.
2. Instagram Stories
Instagram marketing is a great way for small businesses to showcase their products and services. Your business may already be using it as a social media tool, but Instagram is always adding features that you might not be aware of.
Instagram stories are a particularly good way of taking your customers "behind the scenes" of your business and building up a real rapport with your followers.
There are lots of fun features in the stories platform that can make your posts really eye-catching, and it is a great way to live-blog events and product launches.
3. Video Marketing
By 2020 video will account for 79% of all internet traffic. So if your business does not have video marketing strategies, you could be missing out.
YouTube is an excellent platform for product demonstrations, "how to" videos, and even holding Q&A sessions. They can increase brand awareness, and help customers get to know the story behind your business.
4. Email Marketing
Using emails for marketing is not a new idea. However, if you have been using the same old templates for a while, it could be time for your small business to upgrade its email marketing strategy.
Modernize your email content can be a really effective marketing strategy, and demonstrate to your customers that you are a forward-thinking business.
Keep the written content short and snappy, and always include images and videos.
5. Optimize For Mobile
People spend a lot of time looking at their phones. If your small business website is not optimized for mobile, you are putting yourself at an immediate disadvantage.
Always double check all pages of your website to make sure they look as good as they can on all devices. Make sure that your site loads quickly too, as users are less likely to hang around waiting for it if they are on a mobile device.
Tips On Effective Marketing Strategies And So Much More
So those are five tips on effective marketing strategies for small businesses in 2019.
If you found this useful, then why not check out some more of our marketing guides right here.
Things to Consider Before Measuring Digital ROI
Not too sure where to get started when measuring ROI? You're in the right place.
Measuring digital ROI can often prove confusing if you don't know what you are looking for. You can use soft metrics like social media impressions, email engagement, and website visitors which are essential for shaping your digital marketing strategy.
Or you could focus on hard metrics, like spend and revenue. Both hard and soft metrics feed into calculating digital ROI. This confusion often leads to businesses all over the world outsourcing their digital workloads to a digital marketing agency.
First of all, lets lay out some of the types of key performance indicators that can help you determine your ROI –
- Overall digital performance: Traffic, sales leads and user reach.
- Channel: social media platforms, search engines, websites
- Performance based on source: Pay-per-click, sales emails, direct website traffic and organic search.
- Performance based on digital campaign: Conversion rates, lead generation and click-throughs to your website.
Once you have decided which key performance indicators are the most applicable to you and your business, you can then start to measure them to determine your ROI. However, do be aware that measuring ROI does have some disadvantages. It all depends on what type of business you are and what constitutes the return. At the same time, it also depends on what the original investment was. Your company need to determine and agree on both of these factors before measuring ROI.
6 Metrics to Help You Determine Your ROI
Once you gain an understanding of what your ROI objectives are, you need to consider the following metrics, and these are what will ultimately help you determine whether a campaign has been successful or not.
Cost Per Lead. This is the amount that you spend on a campaign which is then divided by the number of new leads you get.
Cost Per Acquisition. This is very similar to cost per lead. The difference is that its an actual transaction.
Click-Through Rate. This metric depends on whether a customer clicks through to your website as a result of digital marketing activity. When calculating click-through rates, make sure that the clicks are directly linked to the campaign you are running. For example, if you are running a paid campaign on Facebook, then any clicks you get from Twitter do not apply to the ROI.
Unique Monthly Visitors. This metric will tell you how visitors are coming to your website every month. However, do be aware that this is a very broad metric to measure and needs more investigating to determine which visitors are a direct result of a marketing campaign. Our tip would be to segment this metric by "paid, organic and social."
Understand Your Customers and Their Consumer Habits
With digital marketing, it's important to recognise the fact that not all customers can be regarded as equal. This is because not all of them will return to spend money with your business on a regular basis. On the flipside of that, some will consume a lot more of your digital marketing efforts but never convert into a paying customer. With this being said, marketing teams need to understand that it's not about the number of online users reached, but the type of user reached.
For example, you need to consider what a customer's transaction value is. You need to identify if they are spending money with you for smaller purchases on an ad-hoc basis or you've have found that they are a consistent buyer and have a loyal relationship with your business. There is another factor that can be taken into consideration and that can be the number of years that a customer has been using your business for. By looking at this will allow you to determine their loyalty.
The best course of action for your digital marketing efforts would be to identify who your most profitable customers are, and where they are coming from in terms of your digital marketing efforts. Did they see a social media post? Or, did they receive a product newsletter from you via email? Returning customers are the ones to have the most positive impact on your business, resulting in repeat transactions and higher profits.
Know Where You Are Spending Your Money
If your digital marketing campaign has been strategically planned and implemented in the right way by creating achievable objectives, then this can be a great way to generate promising results and provide you with data to support any future campaigns. Failing to keep track of how much money has been invested in a digital campaign will cause you nothing but problems and your ROI calculations will be wrong.
Building An Anthill: How to Market Your Pest Control Business
In today's internet-driven world, marketing your small business yourself is easier than ever before. Which, is something that is especially true if you know what you're doing.
People have had problems with pest infestations for as long as civilization has existed. Because of this, you can rest assured that your pest control company will always be in demand.
And, because you know that your customers are already around, the only thing you must worry about is letting them know how to find you.
So, how do you spread the work and track down new customers?
Here's everything you need to know about marketing your pest control business.
Create a Website
As we said before, the internet is the most powerful tool available to small business owners nowadays. And, the easiest way to take advantage of it is by creating a website.
A website will allow customers and clients to get a taste of the services you have to offer, get an idea of how much you charge, and get in contact with you.
While getting any old website up is a good first step, you may want to check out a few other pest control company's sites to get an idea of what you should include.
Check Your Local Listings
Phone books are officially antiquated. These days, if you want to track down a specific service in your area, you turn to something like Google Maps.
If you've been open for a while, there's a good chance you already have an online listing. But that doesn't mean you're in control of the information on it.
You need to claim all your online listings and make sure they include up-to-date contact information and a link to your company's website.
Start a Blog
Once you have your website up and running, you'll need to start generating a little traffic to it. And, one of the most surefire ways to help give your site the SEO boost it may need is by publishing content on it regularly.
A blog will give you an easy way to keep your site's search engine rankings high. While also educating potential clients on pest control issues they may be having.
For example, by publishing an article on what bed bug bites look like, you could alert a new customer to bed bugs in their home and inspire them to hire you.
Get Social
Having a website is an excellent start. But, one of the best ways to build an online presence and track down new customers is setting your business up with a few social media accounts.
Social media can put you in direct contact with your current and potential customers. It also allows you to keep customers up to date on any special deals your offering.
By keeping up with your social media, you can also use it to take control of your online reputation.
Marketing Your Pest Control Business
The internet can make marketing your company a breeze, but only if you know how to harness it. By following these tips, you'll be able to market your pest control business and find dozens of new clients in no time.
Are you looking for a few more tips and tricks to help you take your business to the next level? We can help.
We have dozens of articles to help you start a new business or help you grow your existing company. Check them out today.
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This is Kostia Instagram Profile (@artkostia). Here you can discover all stories, photos, videos posted by Kostia on Instagram Profile.
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"Dill" Congratulations Card
Sale price $4.99 Regular price
FRONT: You're Kind of a Big Dill
INSIDE: Congratulations!
Our mission is showcasing the talents and abilities of individuals experiencing Down syndrome and Autism. This card was designed by Aram, who has autism. You will feel great knowing a portion of YOUR purchase is going to cover the adoption fees of a child with special needs.
Your "Dill" Congratulations order comes with...
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All of our cards are high-end quality you can feel, printed on 14 pt. semigloss, 5x7 paper stock. The designs match the quality of stationary for a card that is perfect for card snobs and everyday use.


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Clean CARFAX. White 2008 Nissan Altima 3.5 SE FWD CVT with Xtronic 3.5L V6 DOHC 24V ******Warranty included*******, Fresh State Inspection and all regular maintenance completed., 3.5L V6 DOHC 24V, CVT with Xtronic.
Checkered Flag Auto Outlet. Value-Priced with vehicles from a Local Business Trusted for over 52 Years. Recent Arrival! 19/26 City/Highway MPG
A 12 month or 12,000 mile warranty is included with every purchase at the Auto Outlet.
* With two doors and a much sportier feel than the sedan, the Altima Coupe is a boldly-styled vehicle for those who want to get noticed. The reasonable price certainly doesn't hurt, either. Source: KBB.com
* The Altima's breadth of powertrain and packaging choices opens the door for lots of comparatively affordable variations on the theme that can meet and exceed the needs of all manner of mid-size sedan buyers -- whether they're seeking basic transportation or want something with a more sporting character. Source: KBB.com
* The 2008 Nissan Altima Hybrid delivers an appealing combination of midsize sedan interior roominess with hybrid fuel efficiency, plus the athletic responses and braking performance that distinguish the Altima among its contemporaries. Source: KBB.com
New vehicle pricing includes all offers and incentives. Tax, Title, License and $699 dealer processing fee not included in vehicle prices shown and must be paid by the purchaser. While great effort is made to ensure the accuracy of the information on this site, errors do occur so please verify information with a customer service rep. This is easily done by calling us at or by visiting us at the dealership.
**With approved credit. Terms may vary. Monthly payments are only estimates derived from the vehicle price with a 72 month term, 5.9% interest and 20% downpayment.
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Why Experiencing Also A lot of Option Is actually Spoiling Your Odds Of Assembly SomeoneAdultBlog dengan No Comments February 21st, 2019 by Noramalina Abdullah.
Another justification it is really most efficient to stick to 1 internet based dating internet page or app at a time, is that when anyone new arrives on to the web courting scene, they sometimes obtain multiple applications and indication about a variety of dating webpages to possess a go searching. Before the appearance for the on-line, relationship was a very many different ballgame.
By continuously turning over the dating wheel, over and over again, hoping to locate anyone far better, you aren't in fact giving on your own any time for you to water and you'll likely end up on this wheel for a particularly long time! Read the rest of this entry »Continue Reading


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Christmas Stocking Charms | Christmas Mouse/Stocking Charms
Metal | Enamel
L 1 5/8'' x W 3/8''
Quantity Per Package: 4 Pieces
WARNING: CHOKING HAZARD Small parts. Not for children under 3 years.
Everything Strawberry Iron-On Patches
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Call Someone Who Gives A Shit Expression Iron-On Patch
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POW Denim Sneakers White Cat Pink Arrows Eye Iron-On Patches
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Heart All Black Everything Iron-On Patch


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When you are renting a 5, 10, 15, 20, 30 or 40 yard dumpster, you want a company you can trust with prices that make you smile. Give us a call today and see the difference we can make in your next construction or clean out project.
Simply give us a call and we will help you figure out your dumpster rental needs.
Our dumpsters usually go out same-day or next-day depending on when you call.
We provide top-notch service, while going easy on your bottom line. What more could you ask for?
Our trained operators are here to give you a fast and hassle-free experience from start to finish.
Once you are done, simply give us a call and we will get your dumpster picked up right away!
Our staff is here to help whenever you need it. Simply pick up the phone and give us a call.
Most cities or municipalities do not have many regulations regarding dumpster rental as long as you keep the dumpster entirely on your own property during the rental period. If you need to place your container on the street in any way, you will likely have to obtain a permit from the appropriate building permit office in your town. You can check with your local public works department to be sure.
Most South Plains dumpster rental companies will take care of securing this permit for you if you rent from them. Make sure that if you're planning to put the dumpster on the street, the company has made the appropriate arrangements. You should also ensure that you get the permit in a timely manner and at the correct cost. If you thought the dumpster company was getting a permit and they did not, you will be the one who will have to pay the fine that is issued by the authorities.
Most significant parts of your clean-up is going to be selecting what's trash and precisely what is not. With out going too crazy, it might be best to err on the side of trashing points, as it's intending to make the job, along with your life, a tremendous amount simpler. Community . can be luring to save all things for foreseeable future "needs," bear in mind you'll get more use through your garage along with your belongings should you have enough area to access these people in the first place.
The web is also a great source of details on these companies. Together with the internet, the homeowners can certainly refine their own search With regards to the region they can be in and they're going to be provided with the name of the companies that offer rentals providers. They will have the opportunity to go through different types of websites and obtain to find the data such as the varieties of containers they provide, the prices as well as the quality of service. A number of them will also be able to use some of the assessment sites of which review the providers of these companies.
Not any property owner or design project are going to be aware of the best industry that can recycle the trash in an successful manner or have enough time to result in the trash reach the industrialist. Disposing the trash to the right place is just a mobile away. You can find number of 5 Yard Dumpster Rental in South Plains, TX companies available online to handle task for an individual.
Dumpster rental can seem such as a difficult aspect to understand. Nonetheless, if these types of simple steps usually are followed, it's rather a very easy and also rewarding method.
Taking the name of industrialization, the western world like US continually run significant projects such as construction, business oriented cleanups, residence renovation. These types of projects produce huge tons of trash that should be cleared quickly, which or else would leave almost all of the counties in the US in dangerous conditions.
When building the home, you will need to check into what size regarding dumpster you actually need. Can the home be considered a smaller challenge thus simply needing some sort of medium sized dumpster? The particular medium sized dumpster hold three tons of debris as well as five pickup truck loads. You might need a large dumpster which will hold 4 tons of particles or 12 pickup truck tons. Most likely you might need a medium as well as large dumpster, not much of a small size dumpster that maintains two tons of debris.
Locating a Reliable Atl Roll off Dumpster Company It is often very simple to identify a company of which deals in Roll off dumpsters in Atl. You can simply try to find them on the net especially in the Atl directory. You'll be able to equally get them through advertising advertisements. A lot of companies dealing in dumpsters usually give prompt distribution and get services with their clients. They've also been available One day online. You could request for their own assistance throughout emergency situations. In relation to rates, many organisations involved in the Throw off Dumpster resources usually offer reasonable prices. Often, the charges add some delivery, pick-up and also disposal of all the so-called waste materials accumulated in the canisters. Reliable companies don't impose separate charges for the functions involved. You could locate them on the net. In order to have a better offer, it's always better to phone the company you would like to deal with early enough. This helps to ensure that you have the proper roll off container that can serve the purpose you are.
It is possible to avoid the additional expense of having to subcontract a specialty cleaning company by having a dumpster located directly on the particular premises in the first place of the challenge. Construction dumpster rentals can be supplied the very next day and is ordered on the phone or over the net. Your business upgrading will look good and work more efficiently when a dumpster can be found on the site prior to a start of construction. At the end of many projects the site is quite dirty, full of dirt, with added materials as well as dirt needing to be found but you could avoid virtually any potential added cleanup with a dumpster set up upon location from the very beginning.
You have now better activities like the property decoration, cooking, inviting buddies over as well as planning out the thrill activities with regard to guests. Pre and post you are completed, you can be remaining will very little or big heaps of trash. Now you don't need to worry about that. There are companies which provide you with quick and disposal trash can and dumpster upon rent to make your work much easier.
Rent any dumpster services typically charge according to a basic price tag and each day fee. You possibly can keep costs down simply by only buying units to get a very specific time frame, nevertheless that can sometimes make items difficult for tasks that over work. Most depots present you with a long term discounted, so make sure you check while booking your unit.
Your den is your haven and you'll do a lot of work in presently there. Once in awhile you could wish you had area for a secure oversized leather-based couch. If you want to remodel your den, as compared to you can do this so that you will are happy together with your den.
Particles collecting trash can or dumpster additionally depends upon company to company. Some offers you secured bins that happen to be when stuffed are captivated by vendor. The packing containers are compiled and taken within presence of a person so that you can take a peek.
If you are on the opposite end of the variety and bought a previously owned home that needs any yard clear, you can also rent any next day dumpster. You could have gotten a deal on a property you want to fix up that you bought due to home foreclosure. Too bad the particular yard is usually a complete chaos and catastrophe. The lawn will not resemble it has been dumped once you get your hands on it once you rent a overnight dumpster.
The important thing to not forget about dumpster rentals is to research before you buy. You don't want to find yourself paying for something you don't need and ultimately won't use.
A final word of advice - be ready for the particular dumpster before that shows up. Clear space on your lawn if that is exactly where you're going to want it set up as well as make sure the control is free you might need where will probably be parked. Should you be ready for your 5 Yard Dumpster Rental in South Plains company to decrease off your trash container, your life as well as the project you happen to be working on will probably be so much easier.
Never procrastinate working with a local dumpster services if your residence is in a essential state. Excessive garbage, trash, in addition to clutter only leads to a lot more filth as well as an unhealthy circumstance. No one in fact wants to stay this way, in particular when there are young children and dogs and cats involved.
Thankfully when you get started on your home design, the next day you may rent a dumpster. To discover which size of dumpster to buy look at the height and width of the construction project you will be taking up.
Every project is made simpler and easier having quick rubbish removal. Cleansing the shed in addition to removing undesired broken devices such as aged rakes and shovels can be done swiftly without having to load the trash by the road in addition to hope that the local rubbish pickup usually takes the entire load. You can grab the guesswork beyond cleaning out your garage, cellar, or drop by eliminating the importance to use local trash collection. Construction tasks are also doable as one dumpster is equipped for old appliances, drywall, bathroom fixtures, garbled metal in addition to broken goblet. Simplify your own home projects simply by calling or perhaps ordering on-line a Md 5 Yard Dumpster Rental in South Plains, TX.
Most homeowners find that 5 Yard Dumpster Rental in South Plains, TX is the best method to take care of the rubbish when undergoing a home redevelopment. This type of services should provide an individual with the pot and, once you are through with the actual container, they may dispose of the trash you have left in it. This service should have a set charge for each sized dumpster they rent out there, but if your going over the fat limit, the charge would be adjusted.
You can clear the backyard, dispose of which trash soon after your residence's renovation and might do a much more for keeping the house clean by using these services. This disposal bin and 5 Yard Dumpster Rental in South Plains, TX companies help you point out bye to individuals heaps of rubbish of your house within a convenient method only.
You may offer provided condos to your condo visitors. You will have to toss the bedroom beds from time period to time. Whenever you rent a dumpster it becomes an easy way to toss large in addition to bulky beds. Any other aged furniture could be thrown out inside the dumpster as well.
Version is one of the recognised processes to home the squander that is generated through tasks like house renovation, design and professional projects. With this process the actual waste material is actually burnt in ashes, but the disadvantage to this process is actually number of un wanted gas like skin tightening and and unsafe gases are released into atmosphere causing very much harm to the surroundings. The effect of the harmful un wanted gas will not be mitigated unless of course the exhaust of un wanted gas into the atmosphere is prevented by using most advanced technology. Nevertheless this method connected with disposing the actual waste is quite expensive and it is advisable only in the countries where there isn't a enough land to dispose the trash.
As far as fees are issue than fees depend upon corporation to company. You'll find many companies which offer their companies on flat charges you could possibly find more costly whereas many companies give the service in rent basis as in you can acquire bin or perhaps dumpster on rent. There are some corporations which provide an individual same companies in more affordable rates however you have to take care while picking these kinds of corporations as there could be hidden fees. So it is advisable that you should go with companies which offer you bin or dumpster in rent. As most of these companies are spread more than many cities so you can find your local service provider. The most important part is that you could save your bin or dumpster carry money which usually many other corporations can ask you for.
Please use the form below to contact us. We look forward to working with you and will get back to you as soon as possible. All fields are required


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Cassie's Reviews > The Miracle of Self Power
The Miracle of Self Power
Cassie's review
Jan 21, 2019
I thought the concept would be great as I'm quite interested in power and its varying dynamic in different situations. The author, however, essentially argues that it's worthless to spend time trying to change anything but yourself (an exceedingly individualistic view) when society shows that people do have influence over how others view a situation. Furthermore, the book brings up religion a lot, which feels very out of place in a book that claims you can only impact and change yourself.
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Reading Progress
January 21, 2019 – Started Reading
January 21, 2019 – Shelved
January 21, 2019 – Shelved as: to-read
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Try to keep social content videos under 30 seconds. Pay attention to aspects unique to social media, like muted autoplay and video looping; you can actually use these tools to your advantage by playing with silent audio and creating seemingly endless loops. Also take advantage of highlighting positive customer feedback in your social videos — this increases your credibility and spreads positive word of mouth.
Cash rules all! Yes, creative planning and strategy are important, but let's be real. Without the proper budget, it's going to be difficult to get exactly what you want. Plan for the money and resources at your disposal. Plan what you'll create or shoot in-house and what you may want to outsource to a production company. Plan what you can splurge on and what you may want to save on.
Using Dr Dave Chaffey's approach, the digital marketing planning (DMP) has three main stages: Opportunity, Strategy and Action. He suggests that any business looking to implement a successful digital marketing strategy must structure their plan by looking at opportunity, strategy and action. This generic strategic approach often has phases of situation review, goal setting, strategy formulation, resource allocation and monitoring.[59]
In a disconnected digital world, people are seeking out ways in which they can feel a sense of engagement and connection. That's one explanation for how authentic storytelling through the use of video is proven to increase engagement. Whether it's through actions like comments, likes, or shares, if people are responding to your video marketing content, that's a great sign that you're on the right track.
But Google has championed machine learning as its future and seems 100 percent focused on growing this segment of the algorithm. Going forward, SEO practitioners will need to be proactive and invest in analyzing data and understanding search behavior across a variety of search and social platforms. This way they can develop the type of content that will motivate and influence user/search behavior which will in turn influence rankings and traffic.
Publishing your video across all your owned channelsExtended ArticleHow to Distribute Your Video on Owned ChannelsIf you know content is king, you probably know distribution is queen. Creating amazing, engaging video content is difficult, but distributing it can be… Read More is probably the easiest way to distribute your video, and the one method that cannot be ignored. It includes every channel you own, like your website, PDFs, digital documents, email lists, ecommerce pages, apps, and more. These channels are the primary sources of information about your company and brand, so use every single one you can intelligently and with purpose.
The combination of charisma, charm and intellect has helped catapult Sharpe to the top of the heap. In a recent conversation with him, I wanted to learn what it truly took to become an expert digital marketer. And one of the most important takeaways from that phone call was that if he could do it, anyone could do it. For someone who failed so devastatingly very early on in life, to rise from the ashes like a phoenix was no easy feat.
Make sure you know what you want your video's call to action to be. You can include your homepage URL, a "Click Here" button, a thumbnail of another video, social icons, or almost any other form of CTA. These are technically graphic elements, but require a little more thought since you'll want to consider what your video goals are and how you want the viewer to accomplish them. Think about their user experience and how you can present your CTA in the least disruptive way possible.
With an agency, you shouldn't worry too much about these details. At Lemonlight, we schedule a creative call to review your ideas and give you feedback based on what we've seen work. We also provide new concepts and any direction you might need. But agreeing on a creative direction is crucial — your creative will be what communicates your branding and what will dictate how memorable your brand video is.
Now comes the script writing, the search for the perfect agency, the video review and edits, and celebrating finally having a beautiful, well-crafted video you can be proud of. The entire production process should take about two months with the right partner, but be sure to plan more time than you need for each of the following individual production stages so you don't fall behind.
Another good news is that your videos don't have to be perfect. It's the content that matters! Latest research shows that users are mostly put off by videos that don't explain the product or service clearly enough. Low quality and poor design didn't matter nearly as much. So it's fair to say that video is like pizza – when it's bad, it's still pretty good!
Tripods range tremendously in price, and the quality of your tripod should depend on the level of camera and lens you have. If you're shooting with your phone, you can get by with a table mount like the Arkon Tripod Mount or a full-size tripod like the Acuvar 50" Aluminum Tripod. For a DSLR, Manfrotto makes a variety of trustworthy tripods starting with the Manfrotto BeFree and increasing in quality and price from there.
Search engine marketing's greatest strength is that it offers advertisers the opportunity to put their ads in front of motivated customers who are ready to buy at the precise moment they're ready to make a purchase. No other advertising medium can do this, which is why search engine marketing is so effective and such an amazingly powerful way to grow your business.
Optimization techniques are highly tuned to the dominant search engines in the target market. The search engines' market shares vary from market to market, as does competition. In 2003, Danny Sullivan stated that Google represented about 75% of all searches.[62] In markets outside the United States, Google's share is often larger, and Google remains the dominant search engine worldwide as of 2007.[63] As of 2006, Google had an 85–90% market share in Germany.[64] While there were hundreds of SEO firms in the US at that time, there were only about five in Germany.[64] As of June 2008, the marketshare of Google in the UK was close to 90% according to Hitwise.[65] That market share is achieved in a number of countries.
Place strategic search phrases on pages. Integrate selected keywords into your website source code and existing content on designated pages. Make sure to apply a suggested guideline of one to three keywords/phrases per content page and add more pages to complete the list. Ensure that related words are used as a natural inclusion of your keywords. It helps the search engines quickly determine what the page is about. A natural approach to this works best. In the past, 100 to 300 words on a page was recommended. Many tests show that pages with 800 to 2,000 words can outperform shorter ones. In the end, the users, the marketplace, content and links will determine the popularity and ranking numbers.
An analytics platform will give you the best overall picture of your important data and where it comes from. The good thing about these platforms are how they're able to tie all your marketing efforts together — your advertising, your social posts, your referral sources, even audience demographics and more. These simplify tracking efforts and put all your data conveniently in one place. There are tons of analytics tools, but here are our favorites.
The majority of new websites created today are built upon WordPress. QuickSprout.com is built on WordPress and most of my readers have site using this popular CMS. I find most people get the basics pretty good for WordPress and SEO, but we're going to take things a bit further in this section. You'll find a detailed walkthrough of setting up the SEO for WordPress plugin, improving your WordPress speed and performance, creating a custom author page and more.
There's a lot to learn when it comes to the internet marketing field in general, and the digital ether of the web is a crowded space filled with one know-it-all after another that wants to sell you the dream. However, what many people fail to do at the start, and something that Sharpe learned along the way, is to actually understand what's going on out there in the digital world and how businesses and e-commerce works in general, before diving in headfirst.
Animated videosExtended ArticleVideo Animation: How to Engage and Captivate Your AudienceYou might know a little about all the different types of videos there are — welcome videos, how-to videos, brand videos, and more —… Read More have the power to engage and captivate your audience like no other. Animation is an interesting style because it can be used independently of the content in your video; almost any type of video can be made in the style of animation. But animation is especially powerful when it comes to explaining difficult-to-understand concepts or technology.Client: Cargopic It's also a great for combining with other styles. You can have a lifestyle video that uses animated graphics or animated cutaways, or you can use it entirely on its own. It's wonderfully versatile and spices up almost any video you create.


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Why the Magna Carta is imporant to students
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Rebel barons, back door dealings and bad King John: in the season of warm fireside stories and ultra camp pantomimes, the Magna Carta makes a rollicking tale. However, for students it should have a particular relevant meaning as so much of the student landscape has been shaped by the Magna Carta - and it should thus act as a source of inspiration. The political engagement of students to say the least is troubled, even more so now that students are not automatically signed up to vote. Something is needed to inspire students, something that is not the normal, sterile and ultimately dull National Union Of Students 'campaigns.' The answer is in the Magna Carta: what better a narrative to inspire anyone than one that makes a King recognise the limits of his authority? Now, the charter itself is considerably more complicated than the story book version of it; indeed the specific details such as church rights and levies on feudal payments might seem a world away. In fact in the short term it was a failure, a document that intended to bring peace but ultimately caused war.
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