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Do you want to build a wooden bed frame stop squeaking, because it doesn't exist in stores?
Do you want to create a wooden bed frame stop squeaking for a specific or custom need?
Having trouble finding detailed plans to build a wooden bed frame stop squeaking?
Find specific step-by-step plans to build a wooden bed frame stop squeaking with descriptions and measurements. Simple instructions for beginners to build a wooden bed frame stop squeaking, no need to be a master craftsman with access to expensive tools and a large workshop!
What if I told you that you could get your hands on 16,000 carpentry projects like a wooden bed frame stop squeaking with plans already "made for you" that include step-by-step plans.
The option of the wood essence is made based on the demands, use and purpose. Sure a common through dovetail joint is a stronger joint compared to a box combined, but it's far more difficult joint to make. A box joint can be cut using a simple jig. The Japanese saw comes in various types, but it always is made up of an easy handle and a tooth blade. It's easy to handle by pulling and cutting, allowing maximum precision. Hardwood boards and softwoods may seem similar in shape and dimension, but they're sold using a completely different measuring system. Softwoods are offered in dimensions, whereas hardwoods are most often offered by the board foot. Beech wood is used in carpentry for the creation of furniture and solid or plywood flooring.
Woodworking Plans PDF for build a wooden bed frame stop squeaking
Would you like to make a wooden bed frame stop squeaking? Whether you are a complete beginner, an amateur carpenter with hand tools or a seasoned pro, you will find thousands of projects that will suit your level and ideas for building a wooden bed frame stop squeaking.
The Japanese saw comes in different forms, but it always consists of an easy handle and a blade. It is simple to deal with by pulling and cuttingedge, allowing maximum precision. Workshop machines like sawing tables, radial, mitre or ring saws are widely used for cutting timber. The radial arm saw can be utilized to crosscut, rip, cut compound angles, dadoes, rabbets and more. As it's a lot of installations and setups to handle all of these tasks that are versatile, the saw has to incorporate a variety of security features as well. Find out how to use it to reduce the probability of injury, before using your saw. Making wooden goods has become accessible to everyone as a result of the development of innovative technologies and ever more efficient tools. Oak timber, among the most prestigious forests, is a tough, homogeneous, solid, resistant, secure and easy to use.
Woodworking is a delicate job that takes a lot of rigour and precision. A design square is a triangular-shaped measuring instrument that has a variety of uses in carpentry and woodworking. Design squares are available in versions, but the 6-inch version is a lot more common. Starting woodworkers and casual DIYers will want one of these helpful tools in the uterus. Wood is a natural, lasting and living material if properly preserved. Wood is a natural, durable and living material if properly maintained. Each of the joinery tools are complementary to each other because a few are used for planing, sawing and cutting while others are used to align the timber.
The fantastic news is that you truly don't have to spend an arm and a leg to purchase a whole bunch of tools to begin. You do not need to have the entire assortment of the ideal carpenter, most of the moment, a few well-chosen tools are enough to accomplish the tasks you've set for yourself. It is ideal to curved or straight precision cuts. Because of the potential adjustment of its speed, it is easy to control and it lets you work in complete security. When preparing to start the saw, the woodworker should check to be sure the blade is spinning freely, rather than participated in the inventory. To make big wood cuts, utilize the electrical saw instead of the manual model since it allows you to cut the timber accurately and with no burrs.
Although hand tools are not always the easiest to use, they're constantly required to complete work or to correct a system. When using a table saw, the suitable zero-clearance blade inserts should be used. When using a stacked dado blade, a proper fit should be utilized. A miter saw is ideal for producing clean crosscuts on wood inventory. You will find variations of the two, such as a miter saw, where the saw motor and blade could be pulled forward a brief distance, a bit. The band saw functions using a closed steel ring. It is a perfect tool for fast and meticulous function, but also for complex shapes. If your stuff are too thick nor too significant, you can get all of shapes, even the most complicated ones. Whenever your materials are extremely thick, work on a desk and with a blade guide if necessary. It is the width of the blade which determines the thickness of the timber it's possible to cut. Before starting any table saw task, assess the security features of your table saw to make sure they're set and working properly.
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Woodworking jigs are useful for creating somewhat difficult tasks easier and consistently repeatable. Most woodworking jigs that may be bought can be homemade. Discover how to create these simple and effective woodworking jigs in such woodworking plans. But, there may not be a more versatile woodworking device. Discover how to get the maximum out of your radial-arm watched these watched hints. Once it comes to woodworking, the exact degree of difficulty depends especially upon the know-how and ability of each individual. Whether a miter saw is a compound miter saw, a sliding miter saw or a fixed motor unit, the rules and things. Whether to get furniture or decoration or to the building of specific building constructions, there are different types of wood which may be worked by means of a timber professional.
When using power tools, wearing the proper safety equipment should be considered compulsory. Garments should be considered, although the woodworker should wear hearing protection and gloves. Another no-brainer tip which doesn't bear mentioning, so that is why I'm going to mention it Always check the inventory you're preparing to cut for any alloy before beginning a cut. Nails and rapidly spinning saw blades are not a fantastic mix. This jig may be used to straighten the advantages of a variety of sizes of planks using nothing more than your table saw. Everyone has probably used a hammer at some point in their lives. The most versatile woodworking version is that the claw hammer having a complete head, while there are many varieties of hammers for a myriad of programs. Pick one that is not too heavy, but feels good. Fortunately, you can protect your saw's table by following these simple tips.
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This jig can be used to straighten the advantages of many different sizes of boards using nothing more than your table saw. A quality tape measure should have both Standard and Metric markings, a locking mechanism and a slightly loose hook on the end of the tape. The hook is loose on its own rivets by design so the user will get results that are accurate if the tape is used to take measurements that are external or internal. When using a table saw, keep the table and surround place free from stock, cutoffs and excess sawdust. Table saws, band saws, and radial-arm saws are cases of woodworking machines which are most often used only in a woodshop because they are much too big and cumbersome to become mobile. Another no-brainer suggestion which doesn't bear mentioning, so that is why I will mention it : Always check the inventory you are planning to cut for almost any metal before beginning a cut. Rapidly and Nails saw blades aren't a fantastic mix.
In most wood stores, the table saw is the centerpiece around which all other work is done. Oftentimes, a radial arm saw can do lots. In this guide, learn about the advantages of a watched, and one can be invaluable in your woodworking shop. The drill press is a very versatile woodworking machine that, due to size issues, is typically only found in the woodshop. In the majority of wood shops, the table saw is the centerpiece around which all other work is done. Oftentimes, a radial arm saw can perform lots. In this article, learn how one can be valuable in your shop, and about the advantages of a saw. Working with wood demands a little skill, but also some tools that are critical for an excellent realization. Distractions are part of normal life, and functioning in the wood shop is no different.
The radial arm saw tends to have a bad rap from many woodworkers, probably since they demand a good bit of space to operate. The radial-arm frequently falls being overlooked for other tools, since woodworkers work out of a garage wood store where space is at a premium. Though the table saw is most likely the workhorse of the woodshop, according to statistics, it's most likely also the most hazardous. A table saw can be quite unforgiving many a woodworker has dropped a finger or worse to this instrument. For little DIY and decorative work, picking wood that is a lot easier to work with will greatly simplify the activities to be performed in woodworking. Ever since woodworking can involve cutting, sawing or grinding, it's thus critical to use blades. With many knots, the wood will be less aesthetic when finished, therefore it's much better to prevent them.


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Senator Murkowski Honors the Great Dr. Martin Luther King, Jr.
Remembering the Life and Legacy of a Remarkable Civil Rights Activist
Today U.S. Senator Lisa Murkowski (R-AK) offered the following reflections on Martin Luther King Jr. Day:
(Click image to view Senator Murkowski's message.)
"Today we pay tribute to the life and teachings of Dr. Martin Luther King, Jr. on what would be his 89th birthday. As we celebrate the legacy of Dr. King, I encourage everyone to take a moment to reflect on all of the good that he did for our culture, our society, and our nation as a whole.
"Dr. King led a courageous fight against prejudice and discrimination, changing hearts and minds across the country. His unwavering mission to bring equality and a more civilized society to the United States is something we must remember through all walks of our lives.
"During his famous speech at the March on Washington in 1963, Dr. King said, 'I have a dream that my four little children will one day live in a nation where they will not be judged by the color of their skin, but by the content of their character.'
"Today and every day, I urge each of us to look to the example he has set; to consider men and women by the content of their character; to learn from the past; and to look forward with hope, supporting one another as we strive to uphold Dr. King's vision of justice, freedom, and human rights for every American."

subset: commoncrawl


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libcinder.orgCinder 0.9.3dev:
Cinder is a peer-reviewed, free, open source C++ library for creative coding.
Please note that Cinder depends on a few submodules. The simplest way to clone it is:
git clone --recursive https://github.com/cinder/Cinder.git
You might also prefer one of our pre-packaged downloads.
Cinder guides and reference documentation are available on the website.
Cinder supports macOS, Windows, Linux, and iOS. It requires Xcode 11.3.1 or later for development on the Mac, and Visual C++ 2019 or later on Windows.
Cinder is released under the Modified BSD License. Please visit our website for more information.
Also be sure to check out the User Forum.


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Hi! Please have the liability waiver filled out and printed for each child/participant before the start of your event!
Make sure to read and sign the event rules/safety regulations and share them with your guests. Please email us with any questions regarding the waiver and our safety rules.
Many thanks!
"Making memories with miniature horses since 2007."- Hayley: Pony Party Bus - Owner, Founder
Thanks for a memorable event season for 2021. We are not taking any additional event bookings at this time.


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Report Highlights Lessons Learned from Texas Hospital during Ebola Crisis
Homeland Security Today. (2015). Report Highlights Lessons Learned from Texas Hospital during Ebola Crisis.
This webpage describes the Expert Panel Report to Texas Health Resources Leadership on the 2014 Ebola Events and presents an overview of the findings and recommendations of an independent expert panel charged with reviewing the circumstances surrounding the first patient diagnosed with Ebola Virus Disease in the United States and two nurses who contracted the disease while treating him.
Login to rate, favorite, and comments on the article


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Location: Inland Village
Price: 150.000,00 EUR
Property Code: HE10171
Object area: 140 m2
Total area: 930 m2
View: Sea view
Stone property on the edge of the picturesque village on Hvar island. The property consists of 3 stone houses and big garden. There are 140m2 of inside space, patio and 2 terraces(one with a sea view). Although it is habitable, property need to be refurbished.
Are You interested in this property?
Please, send us e-mail or call us at 098 177 1136.


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Iconic Medicare Sdn Bhd, IMED is a fully owned subsidiary of Iconic Worldwide Berhad, a public listed company listed in Bursa Malaysia.
Our company and factories are certified under ISO 13485 medical device manufacturer and our products are manufactured with the highest quality standard. Add our products are in compliance with European standard EN14683, CE certified and United State Standard ASTM F2100 for medical grade face mask.
All our products are made in Malaysia and we are registered under Medical Device Act in Malaysia with registration No GA8179120-51201.
Click here to check out our certification.


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Digital transformation is not a one-time action for companies. It demands continuous effort and constant evolution. To be ready for this, companies need to have a strategy in place and a sound technology base, and most importantly, a partner that makes this possible. Using the example of a food service provider, we will show how companies can react quickly and flexibly to new situations.
A New Website is Not Digital Transformation
"Finally, the time has come. The new web store of the food services provider is ready to go live. It's taken a long time to push this through, but now it's done. We have achieved digital transformation! Right?"
Nope! Fictitious example, but a familiar situation.
What does a new e-commerce platform really have? Performance advantages – very likely. But has the digital transformation already been achieved? Certainly not. The next re-launch may already be on your mind. Not simply because technologies are developing faster and faster. Customer expectations are also changing and will continue to change.
A look at China, where the online and offline worlds are already merging completely, shows where the journey can take us. Consumers there can compile their purchases from real goods and virtual offerings in smaller stores while on the move and have them delivered to their homes, on time. Over here in Germany, food retailing may still be in its infancy, but food service providers must prepare themselves for far-reaching changes.
For some time now, large internet platforms and national retail giants have been building up structures. New business models are constantly emerging; delivery services are entering the market, and other criteria are suddenly becoming important alongside freshness and quality. Foodservice providers need to be able to adapt quickly to these rapid changes and challenges – and this is what perpetual digital transformation is all about.
Technology Platforms as Enablers
Modern e-commerce platforms are crucial for digital transformation, but they alone are not the solution. Think of it as a training center for a sports team: players can get in the best possible shape on state-of-the-art fitness equipment; but to win and play to their strengths, they also need the right tactics and team formation. In the world of foodservice providers, these prerequisites are a suitable technology platform, a proprietary strategy, and the right partner.
Companies will have to invest more in IT technology. And not investing may cost the company its existence. Meanwhile, a variety of e-commerce platforms are available that can be adapted and used at a moderate cost. Costs can also be reduced through digitization and automated processing. We recommend cloud platforms and microservices that can be flexibly integrated without interfering with the core system.
Forming the Game Plan: Strategy and Roadmap
So you have the best IT infrastructure and the latest applications. Have you achieved Digital Transformation? Not until you have a clear idea of what you want to do with the technology! What will your business model look like in two years, five years, or ten years?
The starting point for these considerations should be the company's own customers. Whether you're a food wholesaler supplying restaurants and catering services or a retailer supplying end customers, it's always important to put yourself in your customers' shoes. What information does a restaurant operator need for his order? When is the delivery needed? How will the products be used? The supplier that manages to fit its services seamlessly into the processes at the customer's end will ultimately win more customers.
Exploiting the Advantages of the Niche
Data analysis helps understand end customers better. Not only can the customer experience be improved, complementary products or services can also be offered on the basis of order data. Small and medium-sized retailers in particular often have the advantage of the niche. They know their customers in a limited area very well, better than the large players ever could. This is something that must be exploited. Retailers in niche markets can present the products of both large producers and smaller suppliers, to specific customer target groups. And in return, they can ask for support for in-store advertising or online promotions.
Adapting Organization and Processes
Implementing new strategies is never easy. Management may not grasp the profound impact that digital transformation can have on the entire organization. For example, in the food industry, classically positioned companies will have to adapt their internal processes too. Creating a new e-commerce department and recruiting ambitious, well-qualified employees is not enough. The effort to provide customers with the best possible support at every point in their journey would fail if silo thinking of individual departments exists. Conventional rigid structures need to be torn down. The perspective of employees in sales and field service, who may fear for their position, also need to be considered, for them to be open to new ideas.
Now that you know the secret sauce, have a look at how we handled Digital transformation for a Dutch food wholesaler.
When Technology, Strategy, and Organization Interact
Bidfood, a food wholesaler with around 1,700 employees and a good 25,000 customers, knew that their age-old business model had to evolve with the changing times. They wanted to kick off the process in The Netherlands and extend it to their other markets. The crucial questions were: Which customer segment has the greatest potential in the future? What expectations do these customers have? How should processes be adapted to serve these expectations?
With Mindcuv's help, the company gradually eased into a new business model. Instead of the previous large-volume deliveries with low margins, they started to focus more on small, independent restaurants. With a powerful e-commerce platform, the service was noticeably improved, and the range of products was expanded by integrating additional suppliers. When the loss of seemingly secure customer contracts resulted in the loss of almost 30 percent of sales, the company was able to react flexibly thanks to its new strategy. The plans to acquire new customers were implemented earlier, and the company succeeded in acquiring over a thousand new, smaller customers via online channels. The share of online sales increased from 65 to 98 percent.
Digitization eliminated a large part of the administrative workload. The account managers, who used to focus on sales organization and processing, could now concentrate completely on advising their customers in the new environment. The idea of opening up the company's own platform to other suppliers was further developed into a marketplace and one-stop store. With a comprehensive product range, a network of suppliers, differentiated price and delivery models, integration of the latest trends, customers were offered genuine value addition. And the development did not stop there: Currently, the food provider is already working on a brand new project, a start-up.
What does your Future Look like with Digital Transformation?
Driven by accelerated technology development and changing customer expectations, your business environment will continue to change constantly. Perpetual digital transformation requires your willingness to question and change the status quo right from the start. Through this, your companies can react quickly and flexibly to new situations.
Markus Tillmann
Executive Vice President
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Status: Ongoing/Awaiting Decision
Original Run: October 25, 2019 –
Current Seasons: 1
Network(s): Netflix
Genre: Competition, Food
Status for season 2
The show is NOT cancelled, but there is no official status yet on season 2
Series Information:
Nailed It! Spain. On this competition show, novice bakers try to avoid a fiasco while duplicating stunning sweets. At stake are a trophy and 5,000 euros.Oct 10, 2019


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Amazon warehouse jobs in Middletown, Delaware, USA – Apply for Warehouse Team Member Jobs at Amazon – Middletown, DE. See job description, requirements, and the link to apply.
Job Position: Warehouse Team Member
Job ID: 200045528
Job Type: Full time
Required shift times: Overnight, early morning, day, afternoon, weekend
Location: Middletown, DE
Job opportunities vary by location. We update postings daily with open positions.
Hourly pay rate: Earn $15.25/hr – $16.40/hr
Deadline: Immediate openings available now.
Job Description:
Candidates must be 18 years or older with ability to read and speak English for safety.
Reasons you'll love working here:
Health and safety are a top priority with all of our roles and sites. We continue to consult with medical and health experts, and take all recommended precautions in our buildings and stores to keep people healthy.
- Earn more: You can expect a competitive wage and reliable paycheck when you work for Amazon.
- Career development: Many of our entry-level employees become leaders in operations, HR, and other areas. See where your Amazon journey can take you.
- Benefits: Our range of benefits can include health care starting on day one, employee discounts, 401(k) savings plans, paid time off and more! Find out which benefits you'll get after you choose your role with us.
- Stay active: You'll be on the move for your whole shift in our fast-paced environments.
Amazon is hiring for the following types of roles in your area:
Fulfillment Centers – Work inside an Amazon warehouse, selecting, packing and shipping customer orders. If you like a fast-paced, physical position that gets you up and moving, then come help bring orders to life. Work a set, full-time schedule. Shift options include overnight and days, and usually at least one weekend day.
Candidates must be 18 years or older.
Amazon is an Equal Opportunity Employer – Minority / Women / Disability / Veteran / Gender Identity / Sexual Orientation / Age
- Warehouse Team Member Jobs at Amazon – White Marsh, MD
- Seasonal Warehouse Team Member Job at Amazon – Edgewood, MD
- Seasonal Warehouse Team Member Job at Amazon – East Grand Rapids, MI
- Seasonal Warehouse Team Member Job at Amazon – Caledonia, MI
- Warehouse Team Member Job at Amazon – Reading, PS
- Warehouse Team Member Job at Amazon – Gouldsboro, PS
See More: 1,000+ Amazon Warehouse Jobs in USA – Updated Daily
How to Apply
To apply for this job please visit amazon.force.com.


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If your office is busy, then you know just how quickly your space can become cluttered, dusty and dirty if it isn�t cleaned on a regular basis.� Unfortunately, it�s tough to find that balance between having enough time to maintain your office space and having enough time to spend developing and growing the business.� Keeping a clean office is an important part of running a business and the winter months can be a particularly tough time for allergies, colds and flus.� That�s why it�s so important to keep up with the cleaning in order to greatly reduce allergens and help keep those winter allergies, colds and viruses at bay.
By hiring a Sherman Oaks office cleaning team, you can help maintain a dust-free office that everyone can enjoy working in and that is healthy as well.� During the winter months, there are plenty of allergies and viruses that pass through our businesses, schools, churches and other public areas.� If you are tired of dealing with sick employees all winter long, consider how clean your environment is.� Employees who are expected to work in unclean and unsanitary conditions will certainly suffer health problems that could be avoided and their productivity is sure to slide as well.
Dust allergies aren�t the only cause for concern when it comes to keeping an office clean.� There are all sorts of microbes and germs that can greatly affect your health and the health of your employees, but you can�t see them to know when they are there.� As a result, a dirty kitchen, a messy bathroom or dusty desks could be causing employee illness or an increase in allergy symptoms without anyone even realizing it.
Keeping all areas clean and free from dust and dirt is the best way to keep allergy symptoms down and to reduce the passing of viruses among the team. In turn, this helps make employees more productive and helps to keep sick days down to a minimum as well.� For business owners that want to maximize productivity while also taking care of those that work for them, regular office cleaning is a smart and affordable approach to take.


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Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.
Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.
Thermotaxis is employed by mobile animals to select an environment with the most favorable conditions1,2,3. If the climate is excessively hot or cold, this behavior is vital for the survival. In addition, many animals are sensitive to very small differences in temperature in the comfortable range and seek out surroundings with an ideal temperature. This is of particular importance for poikilothermic organisms such as fruit flies, which equilibrate their body temperature with the environment. Assays to monitor larval thermotaxis have been instrumental in identifying and clarifying the roles of molecular sensors such as Drosophila Transient Receptor Potential (TRP) channels4,5,6, rhodopsins7,8, and ionotropic receptor receptors (IRs)9, which endow these animals with temperature sensitivities over different temperature ranges.
A two-way choice test provides one approach to study thermal preferences in larvae6,7. The assay entails establishing two distinct temperature zones and allows the animals to select one side over the other. The results from two-way choice tests can be robust, especially if the temperature differences between the two options are large. In addition, since each assay involves tabulating only two groups, the data can be expressed as a simple preference index. The ease and simplicity of two-way choice assays are also amenable to genetic screens. However, a major limitation is that many experiments are required to establish the preferred temperature of the wild-type or mutant animals.
A gradient assay offers the opportunity to establish the preferred temperature in a single assay8. Moreover, unlike the two-way choice test, it permits the evaluation of the distribution of a group of animals, when confronted with a continuous range of temperatures. One gradient assay uses a Petri dish and single animals and is well-suited for characterizing the detailed behavior of individual animals10. However, since Petri dishes are round, the sizes of the temperature zones vary and are progressively smaller depending on the distance from the center. Therefore, this setup is not ideal for monitoring the temperature selections of populations of animals.
A continuous thermal gradient apparatus that is well-suited to assess the temperature preferences of groups of larvae employs a rectangular arena and is described here. The apparatus is simple to construct and assemble. In addition, the gradient is linear, and is flexible, as it can be used to assess thermotaxis over large temperature ranges from 10 °C to 42 °C. The assay is rapid and straightforward to perform and yields reproducible data. In addition to reporting the favored temperature of larvae, it reveals the preferences of the population of animals over an entire linear range in a single experiment. Due to these advantages, it is an excellent choice for identifying genes required for thermotaxis.
Subscription Required. Please recommend JoVE to your librarian.
1. Equipment Fabrication and Assembling Apparatus for Gradient Assays
- Fabricate the aluminum assay plates for the single-directional gradient assay.
- Trim and grind each aluminum assay plate (Figure 1A) out of a single piece of aluminum using a band saw and sharp vertical mill with the following dimensions: the outer size is 140 x 100 x 9 mm and the inner size is 130 x 90 x 8 mm (Figure 1B). Anodize the inside of each assay plate with black paint to make it easier to visualize the larvae and prevent rust.
NOTE: Fabrication of the aluminum assay plates for the single-directional gradient is done by a machine shop. The anodization is performed by a commercial vendor.
- Mark the upper and lower rims of each assay plate with 13 demarcations, separated by 10 mm using a permanent marker. Place the first and last demarcations 5 mm from the inner edge of the assay plates (Figure 1A, B).
- Trim and grind each aluminum assay plate (Figure 1A) out of a single piece of aluminum using a band saw and sharp vertical mill with the following dimensions: the outer size is 140 x 100 x 9 mm and the inner size is 130 x 90 x 8 mm (Figure 1B). Anodize the inside of each assay plate with black paint to make it easier to visualize the larvae and prevent rust.
- To fabricate the aluminum assay plate for the bidirectional gradient, cut out an aluminum plate with the following dimensions using sheet metal shears: 250 x 220 x 2 mm. Anodize the assay plates with black paint.
NOTE: Fabrication of the aluminum assay plates for the bidirectional gradients is performed by a machine shop. The anodization is done by a commercial vendor.
- Fabricate the aluminum blocks.
NOTE: Two blocks are needed for the single-directional gradient and three blocks are needed for the bidirectional gradient.
- Cut the aluminum blocks (Figure 1C) out of a single piece of aluminum using a band saw (dimensions of 50 x 255 x 14 mm) (Figure 1C, D).
- Drill the water channel inside the block (7 mm in diameter) using a vertical mill and thread grooves in the open ends on one side (Figure 1D). Close the grooved holes with bolts and thread seal tape to form a U-shaped water channel (Figure 1C, D).
NOTE: The fabrication of the aluminum assay blocks is performed by a machine shop.
- Fix the connector in the aluminum block (Figure 1D) with screw threads and thread seal tape at one end. Fit the other end with multiple barbs into silicone tubing (1/4" ID x 3/8" OD x 1/16" wall).
- Obtain two refrigerated/heated circulating baths.
- Assemble temperature-controlled blocks for the gradient assay system.
- For the single-directional gradient, connect each of the two aluminum blocks to a separate water bath so that the temperature of each block is controlled by an independent water circulating system (Figure 2A). Use silicone tubing (1/4" ID x 3/8" OD x 1/16" wall) to connect the outlet of the water bath to the connector on the inner side of the aluminum block (Figure 2A). Also, use tubing to connect the outer side connector on the aluminum block to the inlet of the water bath.
- For the bidirectional gradient, place two of the three blocks under the left and right sides of the plate and connect them to the same water bath with the tubing (Figure 2B). Connect the third aluminum block to a second water bath and place below the center of the plate (Figure 2B).
NOTE: This apparatus and assay are only necessary if the larvae accumulate in the zone at one edge or the other of the single-directional gradient. If so, it is important to assess whether or not there is an edge effect. To test this possibility, establish a bidirectional gradient in which the edge zone temperature preferred by the larvae using the single-directional gradient (e.g. 18 °C) is the temperature in the middle of the plate of the bidirectional gradient (Figure 3E, F).
2. Larval Synchronization
- Nourish flies to be used for egg laying.
- Mix yeast granules and distilled water with a pestle to make yeast paste. Thoroughly grind and stir until the consistency of the paste is similar to peanut butter. Avoid excessively dry paste, which may flake off when transferring flies to fresh vials, or wet paste, which could trap the flies.
- Using a pestle, add the yeast paste close to the inner walls of standard Drosophila vials, just above the surface of the food.
- Count 12-35 females and up to half as many males (but no more than 10) on a CO2 pad, and add the females and males to each yeast paste-containing vial.
- To keep the fly food with the yeast-paste moist, while the flies feed, combine these vials in a tray that holds up to 100 vials with 20 open vials containing distilled water only. Place the tray in a clear plastic bag and seal.
- Incubate the tray in a 25 °C incubator for 48 h so that the females are nourished adequately, thereby enabling them to lay large numbers of eggs.
- Set up vials for egg laying.
- Transfer the nourished flies into new food vials for egg-collecting by tapping them over. Do not use CO2, which may cause the flies to lay fewer eggs over the following short time window.
NOTE: Standard Drosophila food vials are used without yeast paste for egg collection.
- Allow the flies to lay eggs for 3 h at 25 °C so that all eggs are collected over a relatively narrow time window.
NOTE: This will enable the larvae to be synchronized at the same developmental stage.
- Place the vials containing the eggs in the tray containing the water vials and tighten the bag. Incubate at 25 °C under 12 h light:12 h dark cycles.
- Age the larvae for a given number of hours after egg laying (AEL) depending on the larval stage desired.
NOTE: Table 1 is an estimation of the relationship between the hours AEL and the larval stage, which will vary depending on the fly stock, food used, rearing temperature etc. The precise relationship between AEL and developmental stage needs to be verified by each investigator using physical criteria, such as the morphology of the mouth hooks and spiracles11.
- Transfer the nourished flies into new food vials for egg-collecting by tapping them over. Do not use CO2, which may cause the flies to lay fewer eggs over the following short time window.
3. Temperature Gradient Setup
- Prepare single-directional gradient
- To create a humid ambient environment during the assays, place the two aluminum blocks connected to two water baths on wet paper towels, separated by 10 cm (Figure 2A).
- Turn on the two water baths ~2 h prior to initiating the assay to allow enough time for the temperature of the aluminum blocks to equilibrate.
NOTE: It is recommended to perform a mock experiment to determine the temperature of each water bath that is needed to achieve the desired linear temperature gradient. Some typical temperature pairs to set the water baths are listed in Table 2. However, note that the surface temperatures are affected by ambient temperature. The length of tubing also affects the temperatures as there is cooling in the tubes. The length of the tubing in our setup is 1.5 m.
- Microwave 100 mL of 1% agarose at the high-power setting in a 500 mL round wide-mouth bottle and pour 25 mL/assay plate on a level benchtop. Prepare two assay plates, which can be placed on the aluminum blocks at the same time (Figure 2A).
- After the agarose has solidified (10-20 min), gently rub each agarose surface with a standard kitchen sponge or a melamine sponge to make the agarose surface slightly coarse, so that when spraying water on the agarose gel, a smooth thin water membrane will be created, and no water droplets will form.
- Fully submerge the plates in a container with distilled water until the assay is ready to be performed to prevent the plates from getting desiccated.
- Prepare a bidirectional gradient (optional)
- To create a humid ambient environment during the assays, place three aluminum blocks 8 cm apart (Figure 2B) on wet paper towels.
- Turn on the two water baths ~2 h prior to initiating the assay to allow enough time for the temperature of the aluminum blocks to equilibrate.
NOTE: It is recommended to perform a mock experiment to determine the temperature of each water bath for the bidirectional gradient. Some typical temperature pairs to set the water baths are listed in Table 3. However, note that the surface temperatures are affected by ambient temperature. The length of tubing also affects the temperatures as there is cooling in the tubes. The length of the tubing in our setup is 1.5 m.
- To prevent the agarose from spilling out of the plate, wrap the edges of the aluminum plate with labeling tape to form a 10 mm-high wall (Figure 2B).
- Using the high-power setting, microwave 200 mL of 1% agarose in a 500 mL round wide-mouth bottle and pour 120 mL on an assay plate.
- After the agarose has solidified (~30 min), gently rub each agarose surface with a standard kitchen sponge or a melamine sponge to make the agarose surface slightly coarse, so that when spraying water on the agarose gel, a smooth thin water membrane is created and no water droplets form.
- Fully submerge the assay plates in a container with distilled water until the assay is ready to be performed to prevent them from drying out.
- Set up single-directional gradient
- Prepare reagents and items on a bench next to the gradient assay apparatus (see the Table of Materials).
- To promote efficient temperature transfer, fill any gaps between the aluminum blocks and the assay plates by spraying water at the interface between the blocks and plate.
- Using gloves, remove the assay plates from the water container. If water invades between the agarose gel and the plate and causes bumps to form on the surface, remove the water with a P1000 micropipette.
- Place the assay plates on the aluminum blocks so that the demarcations that are 2 cm from either edge exactly match the edges of the aluminum blocks (Figure 2A, C).
- Spray water onto the surface of the plate (a thin water membrane covering the agarose surface is sufficient) to prevent the agarose gel from drying out. Make sure that the water membrane is continuous and free of water droplets since larvae can get trapped in water droplets.
- Cover the gradient system with a cardboard box to reduce water evaporation and help stabilize the temperature of the gel surface. Wait for 5-10 min to allow the temperature to equilibrate.
- Check the surface temperature at 12 points on the plate (Figure 2C). Take two measurements within each zone to establish whether or not there is variability within a zone. Make sure the temperature at both spots is within ± 0.2 °C of the desired temperature.
NOTE: Variability within a zone usually occurs because the exact distances of the two spots to the edge of the aluminum blocks are not identical. Adjust the position of the plate on the aluminum blocks to make sure that the demarcations that are 2 cm from either edge exactly match the edges of the aluminum blocks.
- If the measured temperature gradient deviates from the desired gradient, increase or decrease the water bath temperature setting(s) and recheck the surface temperature after the temperature of the water bath(s) stabilize(s).
- Cover the gradient system with a cardboard box until starting the assay.
- Set up bidirectional gradient (optional)
- Prepare reagents and items on a bench next to the gradient assay apparatus (see the Table of Materials).
- Spray water on the surfaces of the three aluminum blocks to promote efficient temperature transfer from the aluminum blocks to the assay plates by filling any gaps between the surfaces.
- Remove the assay plates carefully from the water container and place on the aluminum blocks. If water invades between the agarose gel and the plate, which might cause bumps to form on the surface, remove the water with a P1000 micropipette.
- Place the assay plate on the aluminum blocks so that the midlines of the first and last zones from either edge exactly match the edges of the two side aluminum blocks (Figure 2B, D).
- Spray water onto the surface of the plate (a thin water membrane covering the agarose surface is sufficient) to prevent the agarose gel from drying out. Make sure that the water membrane is continuous and free of water droplets, since larvae can get trapped in water droplets.
- Cover the gradient system with a cardboard box to reduce water evaporation and help stabilize the temperature of the gel surface. Wait 5-10 min to allow the temperature to equilibrate.
- Check the surface temperature at two points along the midline of each of the 10 zones (Figure 2D). Take two measurements within each zone to establish whether or not there is variability within a zone. Make sure the temperature at both spots is within ± 0.2 °C of the desired temperature.
NOTE: Variability within a zone usually occurs because the exact distances of the two spots to the edge of the aluminum blocks are not identical. Adjust the position of the plate on the aluminum blocks to make sure that the midlines of the first and last zones nearest each edge exactly match the edges of the two side aluminum blocks.
- If the measured temperature gradient deviates from the desired gradient, adjust the water bath temperature setting(s) and recheck the surface temperature after the temperature of the water bath(s) stabilize(s).
- Cover the blocks with a cardboard box until the start of the assay.
4. Larval Collection and Washing
- Isolate clean larvae (option 1)
- Add ~40 mL 18% sucrose solution to a 50 mL test tube. Scoop all the larvae from the food vial(s) with a scoopula and transfer to the 18% sucrose solution.
- Mix thoroughly but gently using a scoopula to separate larvae from the food debris. Wait for 30-60 s until the larvae float to the top layer of the tube. Pour the top layer containing the larvae (~10 mL) to another 50 mL tube and fill the tube with fresh 18% sucrose solution. Wait for 30-60 s until the larvae float to the top layer again.
NOTE: Large food particles sometimes transfer together with larvae and may affect larval thermotaxis if they are not removed. If large food particles remain in the top layer, repeat steps 4.1.1-4.1.2, or manually remove them using a scoopula.
- Transfer the top layer of larvae (~10 mL) to two other 50 mL tubes and fill the two tubes with distilled water to reduce the sucrose concentration, so that larvae quickly sink in water. Wait for 30-60 s until the larvae sink to the bottom. Perform this and the following steps as quickly as possible to prevent the larvae from drowning.
- Discard as much of the water as possible by gently tilting the tubes. Combine larvae into one tube by pouring out the larvae with the remaining water and fill the tube with distilled water.
- Wait for 30-60 s until all larvae sink down and remove as much water as possible by gently tilting the tubes. Repeat this washing step 2-4 times to entirely remove sucrose and all visible food.
- Discard as much of the water as possible and transfer the larvae to an empty 35 mm Petri dish by decanting. Spray the tube with water and use a small paint brush to help transfer the larvae.
- Remove excess water from the Petri dish using a P1000 micropipette. Leave ~0.5 mL of water to prevent dehydration of the larvae.
- Place the lid on the dish to prevent the larvae from escaping. Turn the lid upside down to reduce the ability of larvae to escape through the small gap between the lid and dish. Allow the larvae to recover for 10-20 min.
- Isolate clean larvae (option 2)
NOTE: This alternative method for cleaning larvae mitigates the possibility of suffocating the larvae during the washing procedure. To use this method, proceed with the following steps after performing the above steps 4.1.1-4.1.2.
- Place a cell strainer (300 µm, retains larvae 72 h AEL or older) on top of a 50 mL tube.
- After confirming that there is no adult bodies or food debris floating on the surface of the sucrose solution, pour the top layer containing the larvae through the strainer to trap all the larvae on the mesh screen. Wash the larvae thoroughly with distilled water until the 50 mL tube is filled.
- Remove the cell strainer with the larvae and dispose of the used water from the tube. Place the strainer containing the larvae on top of the empty tube and wash the larvae again with distilled water until the 50 mL tube is filled.
- Turn the strainer upside down over an empty 35 mm Petri dish, and spray water from the top of the cell strainer to transfer the larvae to the Petri dish. Transfer the remaining larvae using a small paint brush.
- Continue to step 4.1.7 above before proceeding to step 5.
5. Assay and Calculation
- Remove the cardboard box and check the gel surface temperature immediately before transferring the larvae to the plate. Minimize the time that the cardboard box is open to prevent disrupting the temperature equilibration. If the surface is dry, spray a small amount of water on the surface.
- Distribute 150 ± 50 larvae near the center of each plate (between zones 3 and 4 out of the 6 zones) for the single-directional gradient (release zone; Figure 2C).
NOTE: For the bidirectional gradient, distribute 200-400 larvae along the middle zone of each half (Figure 2D).
- Place a microplate lid over each assay plate to prevent the larvae from crawling out. Cover the setup with a cardboard box to prevent light exposure, which might affect larval choice on the agarose gel.
NOTE: For the bidirectional gradient, it should not be necessary to cover the plate with a lid, because the plate is larger, and the preferred temperature of the larvae is in the middle zone. Consequently, few larvae accumulate at the edges and have an opportunity to crawl out.
- Allow the assay to proceed for 10-30 min for the single-directional gradient, and for 15-35 min for the bidirectional gradient, depending on the age of the larvae (Table 4).
- Remove the cardboard box and the microplate lid. Photograph the plates from above using a digital camera. Take two photographs of each assay plate so that the investigator can choose the one with better contrast and brightness for analysis.
- Remove all of the larvae from the assay plates and anywhere outside the plates by aspiration.
- Clean the assay plates, the tubes, and the cell strainer thoroughly with distilled water. Reuse the assay plates prepared that day unless the surface of the agarose is damaged.
- Calculate the percentage distribution of larvae in each zone.
- Open the photographic image of the assay results using any software that allows adding markings to the image. To indicate the assay zones, draw vertical lines every 2 cm based on the demarcations on the assay plate.
- Count the number of larvae in each zone and record the numbers. Do not count larvae in regions 0.5 cm from any of the walls. The gel is thicker near the walls, and the surface temperatures are not linear in these regions.
- For the single-directional gradient, calculate the percentage distribution in each zone as follows:
(number of larvae in a given 2-cm zone)/ (total number of larvae in 6 zones) x 100.
- For the bidirectional gradient, calculate the percentage distribution in each zone as follows:
(number of larvae in a given 2-cm zone)/ (total number of larvae in 5 zones on each side of the gradient) x 100.
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Representative Results
To establish an 18 °C-28 °C single-directional gradient, we set the temperatures of two water baths to 16.8 °C and 31 °C. We obtain the temperatures at 13 points by measuring the temperature at 26 positions within the upper and lower portions of all 6 zones, the border lines between the zones, and at the extreme ends of the agarose gel surface (Figure 2C, 2E). The temperature distribution along the gradient was nearly linear (Y = 0.9672*X + 16.19, R2 = 0.9961) (Figure 2E).
We assayed the temperature preferences of control (w1118) larvae at various ages. 1st (24 ± 1.5 h AEL), 2nd (48 ± 1.5 h AEL) and early-3rd larvae (72 ± 1.5 h) showed peaks in the 24 °C zone (Figure 3A, B). The temperature preferences changed during 3rd instar larval development. The largest percentage of mid-3rd instar (96 ± 1.5 h AEL) accumulated in the 18 °C zone (Figure 3A, B), and this bias increased with age. Among late-3rd instar larvae (pre-climbing; 120 ± 1.5 h AEL), ~50% clustered in the 18 °C zone, and the selection of this temperature was ~4-fold higher than the adjacent 20 °C zone (18 °C zone, 50.2%; 20 °C zone, 15.1%; Figure 3A, B). The proclivity to accumulate in the 18 °C zone in w1118 was not due to an edge effect since the late-3rd instar larvae still accumulated in the 18 °C zone, using a bidirectional temperature gradient (Figure 3E, F).
We also tested late-3rd instar larvae (120 ± 1.5 h AEL) with mutations in genes required for discriminating temperature differences in the comfortable range. These include trpA1, which is required for normal temperature selection in the 18 °C-24 °C range5,7,8. Larvae with a null mutation in trpA1 (trpA11) distribute equally over the entire 18 °C—28 °C gradient (Figure 3C). Larvae missing just the A and B isoforms (trpA1-ABG4), or the C and D isoforms (trpA1-CDG4) also show severe impairments (Figure 3C). Flies encode two isoforms of phospholipase Cβ (PLC21C and NORPA), and mutations affecting norpA (norpAP24) but not plc21c (plc21cP319) also disrupt accumulation in the 18 °C range (Figure 3D).
Figure 1. Apparatus for performing the single-directional temperature gradient assay. (A) An aluminum test plate used for assaying larval thermotaxis behavior. The 13 black lines at the top and bottom demarcate 12 zones (10 mm each). The bottom of the plate is anodized with black paint so that it is easier to visualize the larvae. (B) Dimensions of the aluminum plate (indicated in mm). The outer size of the aluminum plate is 140 x 100 x 9 mm. The inner size of the aluminum plate is 130 x 90 x 8 mm. The demarcations are separated by 10 mm. The first and last demarcation is 5 mm from the edges of the inner area of the plate. (C) Top and side views of one of the aluminum blocks used to control the temperature of the gradient. The block has two connectors used to attach to silicon tubes, which connect to a water bath. (D) Dimensions of an aluminum block. The outer size of the aluminum block is 255 x 50 x 14 mm. The diameter of the inner water path is 7 mm. Two 30 mm connectors on the left connect with silicon tubing that extends to the water bath. Please click here to view a larger version of this figure.
Figure 2. Single and bidirectional gradient assay setups. (A) Single-directional gradient setup with two aluminum assay plates on two aluminum blocks. The temperatures of the aluminum blocks are controlled by circulating water from two water baths. (B) The arrangement of the three aluminum blocks, water baths and an aluminum plate (250 x 220 mm) for a bidirectional gradient. The left and right blocks are connected to the same water bath and the middle block is connected to the other water bath. The aluminum assay plate is wrapped with tape to form a 10 mm wall to contain the 1% agarose. (C) Positions to check temperatures (indicated by dots) and to release larvae on the plate. Before initiating an experiment, check the temperature at two points within each zone to confirm that the desired linear temperature gradient is established. Larvae are released within the indicated area near the midline. The larvae are counted within each of the 2-cm zones. (D) Positions to check temperatures (indicated by dots) and the release zones for the larvae on a bidirectional gradient. An equal number of larvae are released along the midline of each half of the bidirectional gradient. The numbers of larvae are counted in each of the 10 (2 cm) zones. One typical set of temperatures (18 °C-26 °C) on the agarose surface is indicated. (E) Temperatures measured along the border lines and midlines of each zone in a sample single-directional gradient. Data represent mean temperatures ± SD. n=8 assays (150 ± 50 larvae/assay). Parts of this figure are reproduced from Sokabe et al.8 with slight modifications. Please click here to view a larger version of this figure.
Figure 3. Representative results using single-directional and bidirectional gradient assays. (A, B) Mean percentages of larvae in 6 zones on the single-directional gradient. Data include 3rd instar larvae at the indicated hours after egg laying (AEL). n=6-7. The error bars in A represent ±SEM. (C) Thermal distributions of the pre-climbing late-3rd instar larvae of control (w1118) and trpA1 mutants on the single-directional gradient. n=3-4. The error bars represent ±SEM. (D) Thermal distributions of the late-3rd instar larvae of control (w1118) and PLCβ mutants on the single-directional gradient. n=4-6. The error bars represent ±SEM. (E) Representative distribution of pre-climbing, late-3rd instar larvae (w1118) on the bidirectional gradient. The left and right assay zones are indicated by dotted lines and separated by the no-count zone in the center (shaded region). (F) Percentage of pre-climbing late-3rd instar larvae (w1118) in each zone along the thermal gradient. Larvae were placed in the left and the right release zones. The assay zones are separated by a 3-cm no-count zone in the center and the distributions are calculated independently. The error bars represent SEMs. n=3 assays (200-400 larvae/assay). Parts of this figure are reproduced from Sokabe et al.8 with slight modifications. Please click here to view a larger version of this figure.
|Hours AEL
|Larval stage
|1st instar
|2nd instar
|Early-3rd instar
|Mid-3rd instar
|Late-3rd instar, just before climbing stage
Table 1. The relationship between the hours after egg laying (AEL) and larval stages.
|Temperature gradient on agarose plate (slope)
|Temperatures of water baths
|Temperatures of aluminum blocks
|10.0-25.0°C (1.5°C/cm)
|18.0-28.0°C (1°C/cm)
|14.0-34.0°C (2°C/cm)
|12.5-42.0°C (2.95°C/cm)
Table 2. Typical temperature gradients and the corresponding temperatures of the water baths and aluminum blocks for single directional gradients.
|Temperature gradient on agarose plate
|Temperatures of water baths
|Temperatures of aluminum blocks
|22-10-22°C (1.5°C/cm)
|26-18-26°C (1°C/cm)
|30-14-30°C (2°C/cm)
|36-12.5-36°C (2.95°C/cm)
Table 3. Typical temperature gradients and the corresponding temperatures of the water baths and aluminum blocks for bidirectional gradients.
|Larval age (AEL)
|Assay time (single directional)
|Assay time (bidirectional)
|24 h
|30 min
|35 min
|48 h
|22 min
|27 min
|72 h
|16 min
|21 min
|96 h
|13 min
|18 min
|120 h
|10 min
|15 min
Table 4. Different larval ages (AEL) and the corresponding assay times.
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To ensure the success of this protocol, it is important to take steps to obtain adequate numbers of larvae to perform the experiments. These include pre-feeding the flies in yeast paste-containing vials for 2-3 d to improve egg laying. The vials need to be placed in a tray containing water vials and enclosed in a clear plastic bag, which maintains the moisture of the food and promotes effective feeding by the larvae while permitting exposure to normal light-dark cycles. However, the yeast paste should not be so soft that the flies become trapped. The number of females per vial depends on the genotype. In the case of w1118, it is usually adequate to allow ~12 females and ~6 males to lay eggs for 3 h. Two vials typically provide enough larvae (100-200 larvae) to place on a plate for the single-directional assay. If the fly stock lays fewer eggs than wild-type or the proportion of larvae that hatch is reduced, add additional females (up to 30-35/vial) and males.
There are many causes of unintended variability when performing these assays. The developmental stage affects the temperature preference of the larvae. Therefore, it is imperative to carefully use larvae of a defined stage. To do so, collect larvae over a narrow time frame (such as 3 h). Since the rearing conditions (temperature, humidity, light-dark cycle and type of food) and some mutations affect the rate of development, do not rely strictly on the time after egg laying to analyze comparably aged larvae. It is also important to examine physical traits, such as the size of the mouth hooks and spiracles7 to determine whether larvae of different genotypes are at the same developmental stage. The times for 1st, 2nd and 3rd instar larvae to develop (Table 1) are based on using corn meal-based food and incubation at 25 °C under 12 h light:12 h dark cycles. Larvae grow slower on molasses-based food. Proper hydration and food freshness also affects the growth of larvae. The amount of water in the food should leave the food neither too dry to peel back from the vial nor too loose due to excessive water. Ideally, the ~5 mm layer nearest the food surface is conditioned by the growing larvae and moves easily when the vial is tilted or tapped. This condition can be achieved using freshly made food with 50-100 larvae.
It is essential to establish a stable temperature gradient before initiating the assay. Therefore, turn on the water bath 1-2 h prior to initiating the assay, as the water baths produce heat and might change the ambient room temperature, which in turn have the potential to affect the gradient. After 1-2 h, the ambient temperature of most air-conditioned rooms equilibrates. However, this must be determined in each environment. In addition, when generating a gradient with a large temperature range (>3.0 °C/cm), it can be difficult to obtain a stable gradient. A tiny movement of the assay plate more significantly changes the temperature gradient when the range is large (e.g. >3.0 °C/cm). We found that a 1-2 °C/cm gradient produces the most stable gradients.
There are several additional considerations that need to be controlled to limit variability in the thermotaxis assays. Washing the larvae is critical, as the presence of food particles or sucrose on the larvae can impact the assay. The washing steps must be performed thoroughly but quickly, because limiting their oxygen supply excessively while they are submerged in water may affect their health. Therefore, we recommend using the cell strainer (option 2) to clean the larvae. A strainer with a pore size of 300 µm works well for larvae at the early-3rd instar stage (72 h AEL) or older. In addition, it is important to perform experiments at the same time of day, such as from Zeitgeber time (ZT) ZT4 to ZT8 (ZT=0 is when lights are turned on), to limit variability due to impacts of circadian rhythms on temperature selection. The level of moisture on the agarose plate can also cause variability in the results. While the surface of the plates needs to be moist, water droplets could trap the larvae, and therefore need to be avoided.
Limiting variability in the assays will make it possible to obtain robust results without performing large numbers of independent experiments. Typically, 3-8 independent experiments are sufficient to obtain a reliable outcome. Proper tabulation of the results is also critical for the success of the thermotaxis assays. Do not count larvae distributed in the regions within 0.5 cm from the aluminum walls, since the temperatures are not linear in these regions. Some larvae will be positioned at the border between two zones, at the time the assay is concluded. If more than 50% of the body length resides in one zone, then include the larva in the tabulation for that zone. If a larva is precisely 50% in each of two zones, then count the animal as 0.5 larvae in each zone.
While the gradient assay allows the larvae to discriminate a range of temperature differences, there are limitations to the lower and upper temperatures that can be effectively tested. It is not feasible to test temperatures <10 °C since the mobility of the larvae decreases greatly at lower temperatures. Although gradients with the upper temperature reaching 42 °C can be established, almost no control larvae stay in any zone >28 °C. Therefore, continuous gradient assays cannot be used to discriminate temperature preferences between various zones >28 °C. However, gradient assays at these elevated temperatures could potentially be used to characterize mutants if they have highly shifted warm temperature preferences.
If a mutant has a locomotor defect, it may be necessary to conduct additional experiments to make sure that the apparent temperature preference is not inaccurate due to a movement impairment. To remedy this potential problem, it may be necessary to establish longer assay times to allow the animals additional time to move to their desired zones. The bidirectional gradient assay can also be employed to test whether the animals select the same preferred zone, regardless as to where they are placed initially on the plate.
In conclusion, the gradient assays described here have advantages over other thermotaxis assays. While two-way choice assays are useful, as they can yield robust results, especially when the temperature differences between the two zones are large, they are not effective in revealing the ideal temperature preferred by the larvae in a single experiment. To do so requires performing many two-way choice combinations to determine the most preferred temperature5,6,7. In contrast, the gradient assay provides the animal an opportunity to select their preferred temperature zone in an environment with a single continuous temperature landscape. Thus, it is unnecessary to devise a large combination of two-way choices to determine the preferred thermal environment. Studying thermotaxis using a round Petri dish has been employed to assess the effects of temperature on the moving dynamics of a larva10. However, it is less useful in discriminating between preferences between different temperatures, since each zone is a different size. Finally, due to the simplicity of the assay, and its utility in discriminating small temperature preferences in a single assay, it can be employed to screen dozens of candidate genes that are potentially involved in larval temperature preference that might otherwise be overlooked using other assays
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The authors have nothing to disclose.
C.M. is supported by funding from the NEI (EY008117, EY010852), NIDCD (DC007864, DC016278) and the NIAID (1DP1AI124453).
|Catalog Number
|Gradient assay apparatus
|PolyScience 9106, Refrigerated/Heated 6L Circulating Bath
|Thomas Scientific
|This model is discontinued. Updated replacement models include: 1186R00 and 1197U04 for 120 V, 60 Hz, or 1184L08 and 1197U04 for 240 V, 50 Hz.
|Aluminum assay plate (for single directional gradient)
|Outer size: 14 x 10.1 x 0.9 cm, inner size: 12.9 x 8.7 x 0.8 cm, black anodized.
|Aluminum plate (for bidirectional gradient)
|25 x 22 x 0.2 cm, black anodized.
|Aluminum block
|Outer size: 25.5 x 5 x 1.4 cm, parameters of inner channels are shown in Figure 1D.
|Connector for aluminum blocks and tubing
|Tygon Sanitary Silicone Tubing
|1/4" ID x 3/8" OD x 1/16" wall
|Catalog Number
|Items and reagents for assay
|USA Scientific
|Pestle for 1.5 mL microcentrifuge tubes
|K type
|Universal microplate lid
|35 mm dish
|Labeling tape (for bidirectional gradient)
|Fisher Scientific
|Fisherbrand labeling tape 2 in x 14 yds
|Prepare 1% solution
|Prepare 18% solution right before starting assay
|Paint brush
|Fisher Scientific
|50 mL centrifuge tubes
|Fisher Scientific
|500 mL round wide-mouth bottle
|Cell strainer (300 mm pore)
|Optional item for larvae washing
|Cardboard box (vial tray)
|Genesee Scientific
|Catalog Number
|Drosophila food
|Distilled water
|22,400 mL
|Cornmeal, yellow (extra fine mesh,flocked) 20 kg
|LabScientific Inc.
|1,609 g
|Brewers yeast 100 lbs
|MP Biomedicals
|379 g
|NutriSoy® Soy Flour (10 kg/unit)
|Genesee Scientific
|221 g
|Drosophila Agar, Type II (5 kg)
|Genesee Scientific
|190 g
|Karo light corn syrup
|1,700 mL
|Methyl 4-hydroxybenzoate (suspend in 200 proof ethanol)
|Sigma Aldrich
|72 g/240 mL
|Propionic acid puriss. p.a.,>99.5% (GC)
|Sigma Aldrich
|108 mL
|Phosphoric acid ACS reagent, ≥85 wt. % in H2O
|Sigma Aldrich
|438081-500 mL
|8.5 mL
- Fowler, M. A., Montell, C. Drosophila TRP channels and animal behavior. Life Sci. 92, 394-403 (2013).
- Palkar, R., Lippoldt, E. K., McKemy, D. D. The molecular and cellular basis of thermosensation in mammals. Curr Opin Neurobiol. 34, 14-19 (2015).
- Vriens, J., Nilius, B., Voets, T. Peripheral thermosensation in mammals. Nat Rev Neurosci. 15 (9), 573-589 (2014).
- Rosenzweig, M., et al. The Drosophila ortholog of vertebrate TRPA1 regulates thermotaxis. Genes Dev. 19, 419-424 (2005).
- Kwon, Y., Shim, H. S., Wang, X., Montell, C. Control of thermotactic behavior via coupling of a TRP channel to a phospholipase C signaling cascade. Nat Neurosci. 11, 871-873 (2008).
- Kwon, Y., Shen, W. L., Shim, H. S., Montell, C. Fine thermotactic discrimination between the optimal and slightly cooler temperatures via a TRPV channel in chordotonal neurons. J Neurosci. 30 (31), 10465-10471 (2010).
- Shen, W. L., et al. Function of rhodopsin in temperature discrimination in Drosophila. Science. 331 (6022), 1333-1336 (2011).
- Sokabe, T., Chen, H. S., Luo, J., Montell, C. A switch in thermal preference in Drosophila larvae depends on multiple rhodopsins. Cell Rep. 17, 336-344 (2016).
- Ni, L., et al. The Ionotropic Receptors IR21a and IR25a mediate cool sensing in Drosophila. Elife. 5, 13254 (2016).
- Luo, L., et al. Navigational decision making in Drosophila thermotaxis. J Neurosci. 30 (12), 4261-4272 (2010).
- Ashburner, M., Golic, K. G., Hawley, R. S. Drosophila: a laboratory handbook. , Cold Spring Harbor Laboratory Press. (2005).


text:
The Coronavirus Aid, Relief, and Economic Security (CARES) Act includes a Higher Education Emergency Relief Fund (HEERF) that provides more than $14 billion in emergency funding to higher education. Of those funds, more than $6 billion must go directly to students in the form of emergency financial aid grants (HEERF-student share) for expenses related to the disruption of campus operations due to the COVID-19 crisis. Bradley University received $2,138,007 in funding for the HEERF-student share.
HEERF-student share dollars are to aid students for expenses related to the disruption of campus operations due to the coronavirus. Note, this is different than a change in a student's financial need due to COVID-19, which could be a result of a loss of income rather than new expenses related to COVID-19.
These FAQs address how students may identify eligibility for funding through the CARES Act and/or how students may apply for emergency financial aid grants for expenses related to the disruption of campus operations due to coronavirus.
A student who is or could be eligible to participate in programs under Section 484 in Title IV of the Higher Education Act of 1965, as amended. A student is eligible if they have filed a Free Application for Federal Student Aid (FAFSA) or if they have not filed a FAFSA but are eligible to file a FAFSA and meets the eligibility requirements under Section 484 in Title IV of the Higher Education Act of 1965.
The only practical way for Bradley University to determine if a student is eligible to participate in the Title IV programs and meet all of the applicable student eligibility requirements is for that student to have filed a 2019-2020 FAFSA. Without having a 2019-2020 FAFSA on file, the university cannot readily verify a student meets the Title IV eligibility criteria and is eligible for an emergency financial aid grant through the CARES Act.
What are the requirements Bradley University has established to award the emergency financial aid grant through the CARES Act?
Bradley University will distribute CARES Act Emergency Relief Fund to students meeting all of the following criteria and who have incurred expenses related to the disruption of campus operations due to COVID-19.
Bradley University will distribute a one-time $400 grant through the CARES Act Emergency Relief Fund to eligible Bradley University students who have incurred expenses related to the disruption of campus operations due to coronavirus. The university will disburse the grant funds directly to the eligible students. Disbursements are not able to be applied to an outstanding balance on the student account. Grant checks will be mailed directly to the student and, by cashing the grant check, the student confirms they have incurred $400 in expenses directly related to the disruption of campus operations due to COVID-19.
Bradley University will consider additional grants to those eligible students who have incurred extraordinary expenses related to the disruption of campus operations due to COVID-19. Eligible students must complete anapplication that will provide the university more details on the student's emergency expenses.
On May 21, 2020, the U.S. Department of Education released new guidance, which broadens the population of eligible students for CARES Act funding. Therefore, the university must now re-evaluate the students who were previously determined to be ineligible to receive grant funding.
With a potential larger population, this also means that the pool of funds available for the application process has decreased. Therefore, the university will distribute the application funds on either a predetermined flat amount or based upon the actual application amount, if the actual application amount is less than the predetermined flat amount.
Clickhere to access the online application. The additional grant funds can be used for expenditures of additional and unexpected costs related to the transition to online learning for the remainder of the semester, including the costs of supplies, technology, etc. that are needed to adequately complete your coursework. Grant funds can also be used for travel or moving expenses to return home due to the transition to online learning for the remainder of the semester; unexpected costs of housing and meals as a result of the campus disruption; and increased medical expenses (including additional support needed for accessibility purposes).
At this time, the maximum amount of additional emergency assistance the university will provide to a student under this application process is $2,600. This amount is over and above the $400 one-time grant. Requested funds do not automatically assure that full or partial funding will be awarded. Funds will be disbursed until all the grant funds are exhausted. If funding is approved the student will be notified by email of the grant amount and the grant funds will be disbursed directly to the student. Disbursements from this fund are not able to be applied to an outstanding balance on a student account.
Based upon the Department of Education directives, International students, undocumented students, and those students in an online program as of March 13 are not eligible to receive an emergency financial aid grant through the CARES Act.
If circumstances are impacting a student's success, Lydia's Promise provides limited emergency financial assistance to current Bradley students who are unable to meet immediate, essential expenses. Designated to off-set a short-term financial need, it is not intended to replace or supplement financial aid. Funds may be subject to federal and/or state taxes. Awards range up to $600. Please visithttps://www.bradley.edu/offices/student/assets/documents/LydiaPromise.pdf for more information.
Will an outstanding balance due to the university limit or prohibit a student from applying for and receiving a CARES Act grant?
No, an outstanding balance will not impact a student's ability to obtain emergency financial aid grants awarded through the CARES Act. The university cannot apply the emergency grant funds to any outstanding charges on students' university accounts. 100% of the grant funds are distributed directly to the student to reimburse the student for expenses incurred that were a direct result of the campus disruption caused by COVID-19. Any outstanding charges are still the responsibility of the student.
While a student does not have to receive financial assistance from Bradley University, the student must be eligible to receive financial assistance from the university.
Yes, a student who was participating in the Hollywood Semester or a semester abroad program as of March 13, 2020, does satisfy this requirement.
Students will be issued a check from the university. The check will be sent to the student's home address. Funds will be disbursed until all the grant funds have been exhausted.
No, funds received through the CARES Act do not need to be repaid. The CARES Act Grant funds are designed to be a grant, not a loan.
No, this fund does not negate nor replace financial aid and is a grant.
No. Emergency financial aid grants under the CARES Act for unexpected expenses, unmet financial need, or expenses related to the disruption of campus operations on account of the COVID-19 pandemic, such as unexpected expenses for food, housing, course materials, technology, health care, or childcare, are qualified disaster relief payments under section 139 of the Internal Revenue Code are not includible in your gross income.
The grant funding will be reflected on the IRS Form 1098T - Tuition Statement that is issued to each student by January 31 of the year following the tax year in which the expenses were paid.
Visit the IRS website https://www.irs.gov/IRSwebsite for more information.
For additional questions regarding the CARES Act funds, please reach out to email@example.com.


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Public tabletop gamer biography
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text: Thanks for looking at some of my published books. The information here will give you basic basic info and links to look at the books at Indiebound (so that you can buy through an independent bookseller) or Amazon. For some of my books, you can find more info and auxiliary materials for teachers here on my site. Specifically, if you're interested in any of the following books, you might want to click on these links to see additional materials: If You Were the Moon, BookSpeak!, the Can Be… books (Leaf, Water, and Rock), and my 30 Painless Classroom Poems set. Cheers!


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Our Founding Fathers Hated Democracy!!!
These United States of America are not democracies, but Republics (each state was actually formed as a separate country, joining together as a Republic Federation. Today, we should have 50 seats in the United Nations, if none at all, which I prefer).
Look at the Article IV Section 4 of our Constitution: "The United States shall guarantee to every State in this Union a Republican Form of Government, and shall protect each of them against Invasion…" Our federal government is not a democracy, but a Republic.
Our founding fathers hated democracy, because it always devolves into some sort of totalitarianism, after going through a socialistic phase, which we seem to be phasing out of at this time.
Mandates are a form of totalitarianism, and we have plenty of those facing us. Do we submit, or do we stand on our constitution? We each may have to face that decision. Do we stand together, or alone? Are you familiar with Aleksandr L Solzhenitsyn, and his Gulag Archipelago? AJM #ACCC4FUSA


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Harnessing the Right Data for Evidence-Based Equity
The Biden administration is determined to improve trust in government, and as part of that goal has made equity a top priority. Agencies are at work responding to recent mandates with assessments about their programs and policies to uncover barriers to serving all demographics and communities.
This collective movement will help rethink longstanding agency operations for program development, implementation, and transparency. But this commitment to evidence-based and continuous improvement requires a thorough examination of the data that will drive our decision making: What information matters most to equity? How can we assess it from the customer's – rather than the agency's – point of view? How do we start to understand and address the root causes of service gaps?
Federal leaders know that improving equity of services for the American people will take sustainable, actionable, and long-term plans to understand what's impacting their customers across their government journey. And they'll need to be equipped with the tools to meaningfully measure their impact along the way.
Here are some core areas of focus for the journey ahead.
Enabling Cross-Agency Insights
It's critically important for each government organization to assess individual service and equity metrics. But the lived experiences of real people include a myriad of interactions that often span different organizations all at once. To reflect this journey, transparency and insights need to be considered from the customer's point of view.
This means being able to look at complex issues, like housing or disaster recovery, and examine the intersections and equity gaps within and across missions. Of course, that clear-eyed picture requires data collected centrally – across divisions – for a transparent and holistic assessment.
Fortunately, we have a blueprint and a foundation for collective reporting. The Digital Accountability and Transparency Act (DATA Act) of 2014 requires agencies to publish spending information on USASpending.gov. With a single data schema and strategy, this unified approach to data collection from different organizations creates a transparent, simplified means of reporting and accessing information. While the DATA Act allows the public to see where funds flowed, we can apply this same unified mindset to answer the question: How do we know if our spending made an impact for more people, and more communities?
The real power of the equity assessments will be in the insights they uncover across the board. If every program assesses its small slice, we can then look horizontally across programs and use the collective data to identify underserved populations, think differently about who's being left out or left behind, share information across programs, and work collaboratively on solutions.
But centralized data collection isn't meaningful if we don't have the right metrics on hand. That gets us to our next issue.
Focusing on Root Causes
When examining data and the flow of funding, it's common to look at the symptoms of inequities. For example, say there's data showing that certain minority small business owners are falling behind on getting contracts. Is it enough to increase the flow of contracts? That's a reasonable decision, but it's just tackling the symptoms and not the underlying causes of the gap. In other words, we need to uncover the "why" behind the data.
As we start to dig deeper, equity requires a focus on "humanizing" data with indicators that are not always quantitative in nature; it can include metrics about respect, inclusion, freedom of identity, empowerment, being heard, equality of opportunity, and accessibility. This information grounds the numbers in the context of lived experiences so we can start to uncover systemic barriers that may be impeding equitable access or outcomes.
Increasingly, we are seeing agencies embrace new methodologies to better understand their diverse customers and populations – which can yield unexpected results and previously unidentified metrics. For example, organizations like the U.S. Department of Agriculture (USDA) are examining areas of improvement through rich ethnographic research and journey maps that explore the intersections and points where collaboration can be improved across programs. Those types of efforts can serve as a roadmap for cross-agency research in the future as we uncover gaps in areas like veteran employment and disaster recovery, where a person needs to engage with multiple agencies at once.
Knowing the Limitations of Data
It's common for agencies to collect customer experience data through surveys, and they're able to use that feedback for short-term service recovery and ongoing improvements to policies, programs, and offerings. But while standard data collection reveals whether services are trusted, impactful, and easy to use – it's just the tip of the iceberg when trying to understand barriers to service delivery.
However, it's very difficult to quantify the part of the iceberg that's under the water to get the whole story. For example, there are the people who:
- Don't know that the services are available or that they qualify for a benefit
- Don't have the proximity, resources, or faculties to access the services
- Don't trust the providers who offer the services
The absence of data about these types of people is in some cases more meaningful than the data on hand. It's important to acknowledge the limitations of data that we have and to navigate ways to address those limitations. Given the restrictions of the digital divide, exploring alternative research methodologies is essential to capture a full picture.
Agencies can partner with state and local governments or community-based institutions to understand, quantify, and fill in gaps around awareness and accessibility. We can also collect this "under the water" data from social listening to help understand customer sentiments and respond to concerns outside of standard survey methodologies.
Following Existing Models for Success
The Home Mortgage Disclosure Act (HMDA) is a notable example of using data to address root causes of an issue and incorporate new measurements of equity into programming and decision making.
The HMDA requires that financial institutions maintain and publicly disclose information about mortgage loans to determine if they are serving communities' housing needs and to identify possible discriminatory lending patterns. While the lending institutions are outside the immediate control of the Federal government, the accountability to report lending data enables the government to track loan approvals across racial demographics, examine root causes of discriminatory patterns, and track equity indicators across a broad ecosystem.
This example about the HMDA – using data outside across the sphere of influence to set meaningful and impactful metrics – highlights the power of collaboration and what's in the realm of possible for the Federal government to effect. So, on a broader scale, how can we modernize for the future of evidence-based equity?
Our colleagues have previously discussed the importance of establishing accountability and shared services to transform the service delivery paradigm for equity. Centralized responsibility would allow for standard procedures, sharing of best practices, and unified infrastructure to collect data from across agencies. It would alleviate the cost and burden of setting up mechanisms in each individual organization and would establish the strong muscle memory required to comply with data privacy regulations, streamline data collection, analyze data sets, and protect the data in storage. And ultimately, it would create sustainability for long-term equity initiatives.
If we can bring that kind of centralized approach to customer experience data – and learn from lighthouse agencies such as the Veterans Affairs' Veterans Experience Office or the USDA's Office of Customer Experience – we'll be able to minimize inefficiencies and gain a more comprehensive understanding of how customers are utilizing the totality of the government's services, address gaps, and better serve all Americans.
Continue the equity conversation by learning more about changing the paradigm to achieve equity in government services, another topic in the Booz Allen series on advancing equity across Federal government programs.
In this series, "Equity as a National Priority: An Interagency Perspective," Booz Allen discusses the topic of advancing equity across Federal government programs – offering perspectives for a framework that prioritizes fair and inclusive service delivery to the public.


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PSYCHO The hardly noticeable pulsation of its heart Beating at the insides like drums; Regular and systematic Going up and down, fascinating. I think I would have watched it still For minutes at length. There was something about it To begin, a rhythm Matching the vein in my head That was about to pop off. The urgency was on me then That vein in my head would burst. A frustrating agony awaits But my eyes refuse to leave the beast. It was drawing me in The study breathing, music. My heart was thumping with the rush But I needed peace, The vein wouldn't stop emphasizing it. I felt the blade of the cool dagger As it drew hot blood gushing; The creature let out a shrill cry And then came silence. The vein was throbbing no more My heart was finally at ease.
FOR THE RATIOCINATOR as I go deeper into the man that he was it seems to explain the previously unexplainable composition of my own character it becomes much clearer in him I find the excuse for my own derangement we share the same deficiency in the configuration of our nature or it's just me unconsciously mimicking the legend probably the later but anyhow it expounds my caricature at least for me and I can no longer deny that I am as much of a lunatic as him a reckless mess trying to mirror his logic the unusual in him calls out to me my insanity is in accordance with his the feverish 'l'appel du vide' that he often claimed to have overtaken him I have, myself, felt many a times before his madness explains mine I blame it on his presence throughout my impressionable years tender age of growth shapes a person's mind mine was made to match his in all of it's abnormality during those vital years of my life I was reading more of him more into him some of the darkness through his words seeped into my soul, unknowingly and still I read him with the intense frequency and adoration of a child till I started morphing into the person he was without ever knowing what exactly I was committing myself to
BLUE It was so empty in that apartment I felt my heart would burst of this loneliness. In that moment I knew, I could never call this place home. Time was running out, It was as if all my life had burnt away Like a cigarette, consumed in its smoke. Just gone. Though I was still here, Still roaming this Earth; Left behind to wander aimlessly. Someone up there had forgotten about me. The wine is turning into vinegar, What a waste! Binging on blue ruin or black smoke, I could still taste blood in the air. The iron assaulting my mouth senseless Meanwhile blue-blooded bastards from under, No good for anything, petty sirens; Were moon kissing their way into oblivion. When I open the windows still, A familiar smell engulfs me. Somewhere down the street, A rose was burning. Can't say I particularly disliked the smell, But it has such a distinct aroma That can be identified anywhere; Smells like innocence on gasoline. It's intoxication feels so wrong, I want to refrain from enjoying it. But I do; One full breath and I am far too deep in it; Right at the bottom of the swimming pool Refusing to swim back up onto the patio, Even if it meant drowning. It's the dark waters that restrain me, You see, but the waves just somehow Romance me into inhaling it; Completely love struck with the poetry. Consciousness makes me feel all mopey So I ditch the norm for a high. Burning with a blue flame, My better judgement, if I had any Couldn't stop me from going on a one way road. It feels like something a sane person would do. And I am so far beyond sane That there can be no scale for it; Guess the burning smell wasn't coming from outside. Did I finally burst a string? Or my ears are just ringing? The past would often hit me, Out of nowhere, like a sledge hammer. Or act like a reminder on the phone That lights up the screen like a flickering light bulb. Yet the future was a beacon of hope for me, One which was continuously moving; Further and further away, So far at last, that it got out of sight. I had officially given up on me; Even when I opened my eyes I saw blue, miles and miles of it; Dark and deep; Dark and deep.
Prachi Kholia is a Master's student at the Department of English and Modern European Languages, University of Lucknow. With a curiosity for everything ranging from Science-Fiction to Ancient History and a passionate love for reading; she is obsessed with the stars and the emptiness they reside in, often trying to weave stories through her poems. Her Instagram handle is _prachi98_.


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This month marks one year from when the pandemic transformed every facet of our work and home lives. Many of you have now been working from home for 12 months with no end in sight. It's very likely you're experiencing "work from home burnout." Being cooped up in one building for all of your personal and work time can start to wear on you. Symptoms of burnout include lack of energy or exhaustion, increased mental distance from one's job, or feelings of negativism or cynicism related to one's career, and reduced professional efficacy. Sound familiar? If so, here are three ways you can combat work from home burnout and get back on track!
Take A Day (Or Two) Off
Even if you have nowhere to go, use up those vacation days! Plan a fun day away from your laptop and zoom meetings, whether you escape to an Airbnb or just a different room of your house. Just because vacations are looking a bit different these days doesn't mean they aren't good for our mental health!
Get Outside
Now that it's March, we're getting subtle hints of good weather. There's no better time to make a commitment to yourself to get outside at least once a day. We recommend even going as far as blocking time off on your calendar to ensure you stick to this one. Take 10 minutes to walk around the block, clean up the yard, or start work on a garden. Engaging in physical activity and soaking up some vitamin D will do wonders for your productivity and attitude!
Block Off Time For "Passion Projects"
We all have day-to-day tasks that are mundane and uninspiring. Instead of letting these responsibilities drive your workday, schedule some time for your "passion projects." We love to kick off the day with these motivating ventures, so block off an hour first thing in the morning. This will get you excited to start each day, and you'll feel productive right off the bat.
Overall, the best thing you can do to avoid work from home burnout is to listen to your intuition. Take breaks when you need them, make time for things that excite you, and communicate with your supervisor and peers. And don't just stop here; for more workplace advice, check out our blog!


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KVD Beauty Metal Crush Eyeshadow
KVD Beauty Tinsel is a slightly cool-toned, light-medium pewter with a sparkle finish. It is a discontinued eyeshadow that retails for $21.00 and contains 0.1 oz.
Jump to a particular section if you know what information you're looking for!
Tinsel is a sparkling, light-medium pewter gold with warmer undertones and a glitter finish. As you might expect, the glitter resulted in a looser, slightly chunkier texture and was the culprit of some fall out over time. For as glittery as it was, though, I think it held on and applied well, and the underlying base color was very pigmented, which helped it look better on the lid over time. It wore for nine hours with some fall out. Clarins Jade #1 (PiP, ) is less shimmery, lighter (90% similar). Tom Ford Beauty TFX1 (LE, $36.00) is darker, warmer (90% similar). ColourPop Snow Flurry (LE, $6.00) is less shimmery, lighter (90% similar). Charlotte Tilbury Seduce Glow (Enhance) (LE, ) is less shimmery, lighter, cooler (90% similar). Chanel Tisse Venitien #3 (PiP, ) is less shimmery, darker (90% similar). JD Glow Fairy Acid (P, $7.50) is more shimmery, darker (90% similar). Chanel Modern Glamour #1 (PiP, ) is less shimmery (90% similar). NARS Silver Screen #1 (PiP, $19.00) is more shimmery, lighter, warmer (90% similar). Dior Constellation (864) #2 (LE, ) is less shimmery (90% similar). ColourPop Ice (LE, $6.00) is lighter, warmer (85% similar). Natasha Denona Glam Green (18M) (PiP, $29.00) is darker, cooler (85% similar). Danessa Myricks Iconic (P, $18.00) is more shimmery, darker, warmer (85% similar). Smashbox Rock Me (PiP, ) is less shimmery (90% similar). LORAC Olivine (LE, $19.00) is warmer (90% similar). KVD Beauty Melancholia (DC, ) is less shimmery, darker, warmer (85% similar). NABLA Cosmetics Gem System (PiP, ) is more shimmery, darker, cooler (85% similar). Sydney Grace John (PiP, $6.25) is more shimmery, lighter, warmer (85% similar). Dose of Colors Slinky (P, $20.00) is darker, warmer (80% similar). MAC Arctic Grey (LE, $17.00) is less shimmery, darker, more muted (80% similar). Bobbi Brown Black Pearl (LE, $38.00) is darker (80% similar). Top 20 dupes listed, see the rest. See comparison swatches / view dupes side-by-side.
DCDiscontinued. $21.00.
Looking for something similar or to see what you have in your stash? Here are some of the top dupes for this product!
Bring a shock of metallic color to eyes. Kat Von D's proprietary formula features Color-Soaked Concentrate™, the result of a technique in which pigment-saturated powders undergo a unique tumbling process that softens and smooths each particle until shadow is creamy and lustrous—leaving behind the highest possible levels of pigment and pearl in this ultra long-wear shadow. With a polished metal finish and 11 striking shades, Metal Crush Eyeshadow glides smoothly across the lids, saturating them with beautiful metallic pigment.


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Put the onions in a food processor and blitz until finely chopped. Put the lamb, lemon zest, spices, parsley and half the onions in a large bowl, and season. Using your hands, mix until well combined, then shape into walnut-sized balls.
Heat the oil in a large flameproof dish, or tagine with a lid, then add the remaining onions, ginger, chilli and saffron. Cook for 5 mins until the onion is softened and starting to colour. Add the lemon juice, stock, tomato purée and olives, then bring to the boil. Add the meatballs, one at a time, then reduce the heat, cover with the lid and cook for 20 mins, turning the meatballs a couple of times.
Remove lid, then add the coriander and lemon wedges, tucking them in between the meatballs. Cook, uncovered, for a further 10 mins until the liquid has reduced and thickened slightly. Serve hot with couscous or fresh crusty bread.