Science Buddies: "Ask an Expert"

I am trying to come up with a natural substance to increasing the availability/activity of the telomerase enzyme. I have found antioxidants that work like vitamin b 12 and folate, I was wondering how I would go about mixing these antioxidants and together in a way where I could try it in worms and see if their telomeres grew?

Hi Jack,

Sounds like you have an ambitious project in mind. Telomere shortening is associated with aging and is a hot area of research right now.

I have some questions, though. What antioxidants are you using and why do you think they will stimulate the activity of telomerase? Did you read this in a published research paper? I know that many people take antioxidant supplements like vitamin C, green tea, turmeric, grape seed extract, etc., but I haven't read that this prevents telomere shortening. Do you have references for this? You need to have an idea of what concentration to use to see an effect.

You said you were going to use worms to test the hypothesis. I assume you mean Caenorhabditis elegans, right? Have you worked with these before? Do you have access to a research lab to work in? How will you determine the length of the telomeres?

Let us know more about the details of your project and we will be able to help you better. Also, how long do you have to work on this project? This is going to take a couple of months probably.

Sybee

This is a class at my high school that I am taking, we have 6 months to work on a research project. I have references to my finding is telomerase research and have thoroughly read enough articles to understand the relationship between telomerase and telomere length. I have done research and found that telomere shortening/attrition is caused a lot from oxidative stress in the cells. That's why antioxidants and stuff would help preserve the telomere length and overall decrease excess telomere attrition. What I found out is that there is a new substance out there called cycloastragenol and is the only know molecule that actually activates telomerase is humans. There are already many "telomere growth and telomerase increasing" products out there. Most of them are very similar in which their ingredients consist of a bunch of antioxidants and vitamins that relieve oxidative stress and preserve telomeres. The number one supplement on the market is TA-65. Which is a patented product that is marketed as the only product that has worked and been tested in humans to increase telomerase activity and overall growth. Ta-65 is largely composed of one ingredient, cycloastragenol. See what I am trying to do is create a mixture of top companies products and their active ingredients, to create the best of both worlds in terms of telomere growth and telomere preservation. Because when people compare these top products they also talk about what the other is missing. But no one ever thinks about combining them or what would happen if you took both of the supplements. So that is what I am trying to do. Create a product that has a good amount of antioxidants and the natural telomerase activator cycloastragenol and give it to C. elegans to see if their telomeres grow or are preserved. I will have a control group and and independent, in which I will be measuring their telomeric DNA as my dependent variable. My issue is, I had contacted a DNA sequencing firm to see if they could sequence my DNA of worms if I gave them it, to observe telomere length and added base pairs. But they told me It would be around 800$ for one sample sequence. So I have to look for another way to measure the worms telomeres. That's one issue- Another issue is actually creating a uniform substance of all my ingredients and actually "giving" it to worms.

I'm not sure I understand why you chose C elegans as the test organism. After it reaches adulthood, there is no more cell division so no telomere shortening can occur. There are different genetic variants of the worms--some have long telomeres, some have short--but this does not affect their lifespan or reproduction:

http://journals.plos.org/plosgenetics/a ... en.0010030

In adult mammals, on the other hand, there are numerous types of cells like stem cells that are continually dividing and thus subject to telomere shortening. I would suggest that you use a culture of normal (not a cancer or immortalized cell like HeLa) human cells like skin fibroblasts that divide and do show telomere shortening. Do you have access to a lab with a biosafety level 2 hood and the necessary tools and reagents to culture human cells?

As to how to measure the length of telomeres without sequencing them, the only way I can think of that you might be able to do it would be to find a restriction endonuclease (https://en.wikipedia.org/wiki/Restriction_enzyme) that cuts in or near the telomeres. You would have to isolate the DNA from the cells, which is easy to do with a kit, cut it with the RE and run it on an agarose gel. The problem then is how to identify which of the bands corresponds to telomeric DNA. Here is one method I found but it requires that you do a Southern blot, which your lab may not have the resources for: https://books.google.com/books?id=h53jl ... el&f=false

If you have access to a thermocycler and the resources to purchase the necessary reagents and primers, there is a method of using polymerase chain reaction, PCR, to measure telomere length: http://nar.oxfordjournals.org/content/30/10/e47.long

I hope this information is helpful. Post again with more questions and we will try to guide you in doing this ambitious and interesting project.

Sybee

Thank you so much for your feedback and suggestions! Unfortunately my school doesn't have the ability to perform cell culture. We can though perform the Southern Blot and PCR process and electrophoresis. Do you think it would be possibly to use fruit flies instead of C. elegans as my test subject for telomere growth? If not, could I just use C. elegans when they are young before they mature to adults? Thanks, Jack.

Hi Jack,

Sorry to hear that you can't do tissue culture. Doing your experiment with human cells would be the simplest and best method.

As to which would be better, immature C elegans or fruit flies, I really don't know. I don't really know how many cell divisions it takes to cause a noticeable shortening of telomeres in those organisms. The worms would be easier to expose to the antioxidants and telomerase activator than the flies, but the flies live longer and may show more telomere shortening. Look up some journal articles on fruit flies and see if any telomere research has been done using them. Also check telomere formation in developing C elegans to see if you might be able to use them.

Also, in order to identify the telomeres, you will need a specific DNA probe for the Southern blot or a set of primers for the PCR. You'll have to do some more research to come up with specific probes or primers for telomeres in the worms or the flies and I can help you do that. They can be fairly expensive to synthesize, but there are some companies that offer smaller amounts for researchers to test. If you are going to do a Southern blot you will also need to have a tag added to the probe so it will show up on the agarose gel. Here's a company that supplies one type: http://www.biosyn.com/hrp-oligonucleotide-probes.aspx

Here's another reference for nonradioactive DNA probe labeling and Southern blotting: http://www.dartmouth.edu/~staphy/protoc ... probe.html

Good luck!

Sybee

Thanks for the response! I am actually thinking about going down the path and using yeast instead of fruit flies and C. elegans to test for telomere growth. They seem easier to work with and are already sequenced. They also divide really quickly and are easy to replicate. Would you be able to come up with a way to extract the DNA, method for running PCR as well as performing a blotting technique? Thanks, Jack.

Hi Jack,

Yeah, I agree that yeast is easier to work with than fruit flies or worms. Just be sure when you write up your report that you research the differences between telomerase in fungus and telomerase in mammals. You want your conclusions to apply to higher organisms so you need to find out if the telomere systems of a yeast differ in any substantial ways from those of mammals.

Isolating DNA from yeast is pretty simple. Does the lab you plan to work in have a standard procedure for isolating DNA from cells? If they do then you should use that. Here's a simple procedure for isolating genomic DNA from yeast: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182553/

Your lab should have the necessary chemicals and equipment to do this procedure and it will give you enough DNA to do the PCR and the Southern blot.

You'll have to decide what concentrations of test substances to use and I would do this by adding a range of concentrations to the yeast cultures and measuring the growth rate. You don't want your treatments to kill the yeast! Hopefully your lab has a hemocytometer which is a special microscope slide that is used to count cells. If they don't have a hemocytometer you could determine growth by using a spectrophotometer or nephelometer that can measure the turbidity of the cultures. If you don't have any of these instruments then you will just have to judge growth by eye. Put the cultures side by side and photograph them so you have a record of what they looked like after the treatment.

The next question is what set of primers to use for PCR so that you can determine the length of the telomeres. I don't know what they would be and you'll have to do some serious reading and talking to scientists who can help you. When I do PCR I am looking to see if a specific sequence is present, not to measure the length of a region of DNA. I don't know how familiar you are with how the polymerase chain reaction works but it requires two primers, one forward and one reverse. In order to choose these you need to know the sequence of the DNA that you are trying to amplify. The problem I see with measuring the length of a telomere by PCR is that the ending sequence is going to depend on how much has been cleaved off--how short the telomere has become. Naturally this will be different for different telomeres. So what reverse primer would you use? Maybe one of the other experts can answer this one or the scientists from your lab.

Doing a Southern blot may be the best way to measure telomere length. All you would need is a tagged DNA probe that is complementary to a specific region in the yeast telomeres and a way to cleave the telomeres away from the rest of the DNA at a point close to where the telomeres join the chromosomes. DNA cleavage is done using enzymes called restriction endonucleases and New England Biolabs has a program where you can plug in a DNA sequence and it will tell you what enzymes will cut that sequence: https://www.neb.com/tools-and-resources ... yme-finder

I hope I am not hitting you with too much information all at once. Molecular biology is college level science and I know a lot of this stuff is unfamiliar to you, but it really is not difficult to understand. Youtube has a lot of good videos on isolating DNA, doing Southern blots, selecting probes, doing restriction digests, etc., and you can learn a lot by watching those. Make a list of the questions you have and we will help you.

Good luck!

Sybee

Thank you for the feedback once again!I will continue to research the connection between the telomere systems in yeast and mammals. I will also look for the set of primers that I will to perform PCR with. I will be ordering the ingredients for my substance soon and will reply back to you with any questions that I have. Thanks, Jack.

You're welcome! Good luck finding all the information. Don't hesitate to call professors or grad students at universities. They are happy to help young students with questions about methods and the science behind them.

If you get stuck on anything, be sure to send us a post. We want you to understand why you do things in certain ways and not just follow a recipe or formula. You learn a lot more that way and can answer the judges' questions much better.

Sybee

Hey! What do you think about instead of measuring the telomere length via PCR and Southern Blot, I instead just measure the amount of times the yeast replicate as my dependent variable. I figured that would be justifiable for the efficacy of my product, because that would show which group the exp. or control can replicate longer, thus who has longer telomeres. This would be a ton simpler to do in the lab, and would totally be manageable. Thanks, Jack.

You could just measure yeast cell numbers as your dependent variable, but this would not prove that your treatment prevented telomere shortening. The treatment could stimulate cell division in some other way. I'm afraid the only way you can say that a substance keeps telomeres intact is by measuring their length.

If you do decide to use cell growth as your outcome you would want to measure the rate of growth as well as the number of cells at the endpoint. That would mean that you have to count the number of cells at specific time intervals and plot the number vs time. If the slope of the line for the treated cells is the same as for the non-treated controls then you can say that the treatment does not increase the rate of growth. Keep counting the cell numbers until they stop increasing and start to decline. If your treated cultures grow at the same rate as the controls but reach a higher final number then you could say that this might be due to telomere preservation.

If you have additional questions, please let us know.

Sybee

Thank you, I understand what you are saying about my end conclusions and will get back to you with any questions regarding my project soon. Jack

Hey! I was wondering if I could get your insight on what the simplest procedure would be to go about my research? Thanks, Jack

Well, if you want simple just grow some yeast, treat it with antioxidants and see if those cultures produce more cells than a control without antioxidants. This experiment won't tell you whether telomeres in yeast treated with antioxidants are longer than those without, but you could test several different antioxidants and predict which one works best as a hypothesis.

If you want to measure telomeres you will have to do the experiments that I already mentioned.

Sybee

Thank you! I will take what you said in consideration as I plan my procedure.

I would advise you to run your procedures by us before you finalize them. We are experts in planning and developing workable projects and we may be able to suggest some modifications to improve an experiment or ways to avoid problems.

Good luck!

Sybee

As soon as I formalize my procedure, I will run it back to you guys!

OK, Jack. Good luck!

Sybee

Hey! I am currently creating the homogeneous substance that I will use for my experimentation. I am having an issue with understanding how much of each ingredient I should be using. The problem is I don't want to kill the yeast when I mix it into the agar medium. I need to figure the right quantity of each ingredient to use before I do my experimentation. Do you think I should just use the trial and error method and mix certain quantities of my substances with my yeast and see what happens? Also I am currently looking for mentors for this class. It is a requirement and is necessary for when we write our research proposal and the actual paper. I am currently not getting any responses from the emails I am sending out to the different college's professors. I was wondering if you would want to be my mentor and be listed on my abstract and etc. It would entail frequent email communication like we have been doing already. Let me know if you would be interested!

Thanks once again, Jack

Hi Jack,

I think for a high school science project that the requirement is for a mentor that you can talk to in person. Don't use email. It often ends up in the spam folder or is ignored. I would find a nearby college or university and make some actual phone calls. Before you call, however, think about exactly what you want to say so you can give your proposal in the briefest time but with all the necessary information. Aim for about 3 minutes, and you must state your case very clearly and persuasively. They will want to know exactly what their responsibilities are and how much time it is likely to take.

Now the question is whom should you call. Since your hypothesis is that certain antioxidants will preserve telomeres, you should look for professors in biomedical sciences such as at a medical school or in the biology department. Professors usually have web pages where they describe their research interests and you should read these to find out those individuals who would be most likely to be interested in the role of telomeres in aging or cancer. Once you find some good candidates you should call them directly and politely ask them if they or someone in their lab like a graduate student or lab tech would be able to act as your mentor. Tell the person that this is a requirement of your high school and that you would be very grateful for the help. Be very clear about the responsibilities of your mentor like whether you have to meet with them in person, how often you will need to communicate with them and that their name will be associated with your project.

If you still can't get someone to agree to be your mentor then of course I will do it, but first ask your teacher if the requirement can be met by using an online science project service like Scibuddies. The rules may say that the mentor must be someone at a university and you may actually have to talk to them in person.

In regard to your initial question about how much of your preparation to use I was a little confused by what you said about adding it to agar. How did you plan to determine the numbers of yeast cells in the treated cultures compared to controls? The usual way to test a substance's affect on cells quantitatively is to do a liquid culture. You would count the number of cells in the culture using a hemocytometer at the beginning and again at the end to see if the antioxidants allowed the yeast to divide more times before stopping. Instead of counting cells using a hemocytometer, you could plate them on agar and count the colonies but you would not have the antioxidant present in the agar. Can you clarify your reasoning in this experiment?

Sybee

Thanks for the response! I am currently reaching out to local universities and colleges in search of a expert related to my area of research. I am allowed to have a mentor online. You have been very helpful to my project so far and I would love to continue to collaborate with you. Have you written any published articles?

I have to decided to use solid agar embedded with the various antioxidant substances as my substrate. I was planning on counting the yeast cells with my naked eye. I see what your saying about the liquid culture. My control group is the agar without my substance added to it. My experimental group will be the agar with the substance added to it. Based of the rate of growth and amount cells replicated I will be state a correlation between my substance's presence and telomere growth. The longer the telomeres are the longer the cell can divide. Let me know what you think?

Scientists need to measure things accurately. I know a project becomes more difficult when you have to make measurements that generate actual numbers but that is the only way you can test a hypothesis. You need to count the numbers of yeast cells, as I said. If you plate them on agar containing some concentration of your test substance and on agar alone you will get the same number of colonies in both cases, provided that you spread the same amount of yeast on each plate. What will you be looking for? Larger colonies on the plate that has the test substance compared to the plate that does not? You can do it that way and maybe you will see a difference, but it won't be quantitative and you won't be able to do a statistical test to prove or disprove your hypothesis.

The experiment can be easily done as I described it to you. Make liquid cultures of the yeast and to one set add different amounts of your antioxidant mix. The other set gets only water. Let the yeast grow at 27-32C and do cell counts every day. If you can't borrow a hemocytometer from someone then just plate the yeast on nutrient agar and count the colonies that form. This will tell you two things--the rate at which the yeast are dividing and how long they continue to divide. Those are the two necessary measurements you need to test your hypothesis that the antioxidants promote longevity.

I hope it is clear to you why you need to make quantitative measurements in an experiment. If you are not sure about something please ask us. If you need help with the methods, explain the procedure to your teacher and she/he should be able to give you advice. Otherwise ask us and we will give you detailed instructions. Science Buddies is your mentor and any expert can offer advice on your project.

Sybee

Thank you so much for the response! I am setting up my experiment and I am taking your suggestions into consideration. I am starting with placing yeast on solid nutrient agar and then several days later, I will compare the differences between the amount of yeast cells between the control and experimental groups. I am going to do the liquid culture in quantitative measurements as you suggested.

In regards to the mentor ship, I am required to have forms signed by the mentor who is guiding my project. Would you be willing to sign this form?

Thanks, Jack.

Hi Jack,

Good job on setting up your experiments! Let us know as soon as you get some results. We always like to know how a project turns out so we can offer better advice to other students.

Experts must remain anonymous and cannot contact students off the Science Buddies platform. That is the rule that applies to all experts. Explain to your teacher that you are being mentored by an expert forum associated with an international online science project help service. If he/she needs proof just give them the link to the website.

Good luck!

Sybee

Thanks, I will keep in touch as my experiments progresses!

OK, Jack. Remember, we are still your mentors so if you have any questions at all, don't hesitate to ask.

Good luck!

Sybee

Sounds good!
Thanks, Jack