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Hamden Hall  Country  Day  School     Journal  for  High  School  Research   Volume  I          


Spring,  2012      

Number II  

Projects     The  Effect  of  Resveratrol  on  the  Metamorphosis   Rate  of  Vanessa  Cardui   -­‐  Isaac  J.  Katz-­‐Justice,  2015  


The Effect  of  Varying  Concentrations  of  Caffeine  on   the  Heart  Rate  of  Daphnia  Magna   -­‐  Josie  L.  Maynard,  2015  

Papers The  Prevalence  and  Zoospore  Load  of  Batrachochytrium  Dendrobatidis     Distributed  Across  Northeastern  America   -­‐  Jack  R.  Dowe,  2013     UVB-­‐Induced  Psoralen  Photoadducts  and  Their  Rapid  Detection     by  Surface-­‐Enhanced  Laser  Desorption  Ionization  Time     of  Flight  (SELDI-­‐TOF  Mass  Spectrometry   -­‐  Alexandru  D.  Buhimschi,  2013  


Isaac Katz-Justice*, Dr. D’Angelo*

Department of Biology, Hamden Hall Country Day School*

Correspondence and reprint requests to:

Dr. D’Angelo Department of Biology Hamden Hall Country Day School 1108 Whitney Avenue Hamden, Connecticut 06517 Phone: 203-752-2600 Fax: 203-752-2651 e-mail:

I. INTRODUCTION Resveratrol is a natural phytoalexin (an antimicrobial toxin produced by plants) that is found in the skin of grapes and Japanese knotweed (Fallopia japonica). Resveratrol has been shown to increase the longevity of both invertebrates and vertebrates by activating the gene that controls the production of the protein Sir2 in invertebrates, and SIRT1 in vertebrates. Resveratrol has been shown to halt mitosis in cells at the S/G2 phase transition. Mitosis, the process of cell division, plays a crucial role in insect metamorphosis. The purpose of this experiment was to determine whether adding resveratrol to the diet of Painted Lady (Vanessa cardui) caterpillars would affect the rate of metamorphosis. The proposed hypothesis was as follows: If resveratrol is added to the diet of Painted Lady (Vanessa cardui) caterpillars, it will take longer to complete metamorphosis, due to the disruptive effect resveratrol has on cell mitotic activity. My independent variable was the diet of the caterpillars (whether or not they had been fed resveratrol) and my dependent variable was the length of metamorphosis. Due to time constraints, our experimental objective was modified. The revised hypothesis looked at how long it took caterpillars to enter metamorphosis, not how long it took for them to complete it. The lifespan of a Painted Lady caterpillar can be divided into four stages: the egg stage, the larval stage, the chrysalis (or pupa) stage, and the adult (or reproductive) stage. This experiment is focusing on the transition from the larval to pupa stage. In this paper, the words metamorphosis, chrysalis, and pupa are used frequently. Metamorphosis describes the process of the change from a caterpillar to a butterfly, chrysalis refers to both the process and the cocoon, and pupa refers to the cocoon only. The pupa stage,

found only in organisms undergoing complete metamorphosis, is classified as the stage where the organism is undergoing transformation. Sir2 and SIRT1 are members of the Sirtuin family of proteins. Consisting of 7 proteins, Sirtuins are used by the body to regulate metabolism (metabolism being defined as the set of all actions taken by a living cell in order to support life). So far, there have been only two events found that signal the body to produce Sirtuins: starvation and the ingestion of Resveratrol. When Sirtuins are produced, the cells in the body go into a “defensive� mode. This mode lengthens the life cycle of a cell, as well as halts mitosis. When present, Sirtuins have been shown to lower blood sugar levels, and increase longevity of the organisms present. Because of these health benefits, Resveratrol is of extreme interest to scientists, and is an active area of research.


33 Vanessa cardui caterpillars.

15 150ml Liquid Resveratrol pills

462 grams of butterfly nutrient

33 plastic cups, with lids 6 large plastic containers, with lids

B. Method 1. 33 larval Painted Lady caterpillars were purchased from 2. The caterpillars were placed into identical plastic cups, with 14 grams of butterfly nutrient. 3. The caterpillars were split into 2 groups: A control group consisting of 18 caterpillars, and an experimental group consisting of 15 caterpillars. The control group was then further split into 3 groups of 6, and the experimental group was split into 3 groups of 5. (See Figure 1) 4. One 150milliliter Resveratrol pill was allocated for each of the caterpillars in the experimental group, and was mixed in with the 14grams of butterfly nutrient. 5. The caterpillars remained in their cups until two days after entering metamorphosis, at which point they were moved into the large plastic enclosure. The experiment lasted 3 weeks (21 days) From Thursday April 5th until Thursday April 26th. The caterpillars required no active maintenance during their larval stage. While the actual experiment involved quantitative data as to how many caterpillars had entered metamorphosis after a given period of time, qualitative data as to the health of

the caterpillars, as measured by size and activity (see Figure 2) was recorded every day. It is important to note that, because a latticework of webbing was being weaved, the caterpillars could not be removed from the cups in the larval stage in order to be measured. Thus only rough measurements could be taken.

Figure 1: The Experimental setup. (The Control Group is on the left.)

Figure 2: A comparison between a Control caterpillar (Left) and Experimental caterpillar (Right).

III. RESULTS The data shows that Resveratrol had an effect on the caterpillars’ metamorphosis. 10 out of the 18, or 55.56%, of the control caterpillars had entered metamorphosis after three weeks, while only 3 of the 15, or 20%, of the experimental group caterpillars had. Otherwise put, for every caterpillar in the experimental group that entered metamorphosis there were 2.78 caterpillars in the control group that had entered metamorphosis. The raw data from this experiment can be found in the lab notebook attached to this lab report. Table 1: Data Collection Table Control 1 Dead 5 Alive, in larval 0 stage Alive, in 1 metamorphosis

Control 2

Control 3 0 2

Experimental 1 2 0

Experimental 2 1 4

Experimental 3 3 2

0 1 5





Table 2: Data Averages Dead Alive, in larval stage Alive, in metamorphosis

Entire Control Group 5 3 10

Entire Experimental Group 6 6 3

Graph 1: A Bar Graph Showing The Number of Caterpillars Dead, Larval, and Metamorphosing After a Period of 3 Weeks.

The Percent  of  Caterpillars  Dead,  in  the   Larval  Stage,  and  in  the  Pupa  Stage  after  3   Weeks.   100%  



Percent of  caterpillars  in  each  state  



50% Pupa   40%  

Larval Dead  






IV. DISCUSSION The results support the hypothesis that if you add resveratrol to the diet of a Painted Lady caterpillar (Vanessa cardui) then it will take longer for that caterpillar to enter metamorphosis. The data illustrated that, after 3 weeks, only one third as many caterpillars that have taken resveratrol entered metamorphosis as caterpillars without resveratrol in their diet (Data Table 2). In exact terms, a caterpillar that has not ingested resveratrol is 278% more likely to enter metamorphosis in a given period of time than a caterpillar that has ingested resveratrol (Graph 1), a huge disparity. The data corroborates that resveratrol has a marked effect on the life cycle of invertebrates, and would appear to suggest that because the caterpillars fed resveratrol entered the chrysalis stage later than those caterpillars not fed resveratrol, they will in fact have a longer lifespan. The growth of the experimental caterpillars is stunted compared to that of the control group (Figure 2), which is logical because of resveratrol’s disruptive effect on cell mitosis, a necessary part of biological growth. Resveratrol is a mild toxin, and it is possible that the dose given (150ml) was large enough to harm the caterpillars, which could explain both the stunted growth and the late metamorphosis. Other experimental errors include the fact that light dosages were not kept constant for all caterpillars, infections may have arisen due to possible food contamination, the air hole provided could have been too small to let in the necessary amount of oxygen, and the caterpillars could have been chosen with an unconscious bias so that the healthier ones were put in the control group. Future studies should be designed to ensure that the data collector is blinded to the group classification of the caterpillars. The biggest weakness in this experiment was the timeframe, which did not allow enough time to study the length of time needed to complete metamorphosis, which was the original goal of the experiment. The first caterpillar to start metamorphosis did so 14 days into the experiment, with

only 10 days left. I believe the ideal timeframe for this experiment would be 3-4 months, which would not only give the opportunity to observe the butterflies entire life cycle, but would also, assuming the caterpillars treated with resveratrol could reproduce, allow for the study of resveratrol’s effects on the subsequent generation. Moving forward, I would like to look at the genome of the caterpillars treated with resveratrol to see how it differs from the genomes of untreated caterpillars. Because resveratrol activates a gene, it should leave an altered genome behind it, and the gene that controls the production of Sirtuins could hold the key to unlocking the secret of extreme life longevity for humans.

VI. LITERATURE CITED 1. Lagouge et al., Resveratrol Improves Mitochondrial Function and Protects against Metabolic Disease by Activating SIRT1 and PGC-1a, Cell (2006), doi:10.1016/j.cell.2006.11.013. 2. Ragione, FD et al., Resveratrol arrests the cell division cycle at S/G2 phase transition., n.p., 8 Sept 1998, Wed. 18 April 2012, 3. Valenzano, Dario R., et al. Resveratrol Prolongs Lifespan and Retards the onset of AgeRelated Markers in a Short Lived Vertebrate. N.p., n.d. Web. 11 Apr. 2012. 4. Van der Kloot, William G. Insect Metamorphosis and Its Endocrine Control. Oxford University Press, Feb. 1961. Web. 18 Apr. 2012. <>.


Josie Maynard*, Dr. D’Angelo*

Department of Biology, Hamden Hall Country Day School*

Correspondence and reprint requests to: Dr. D’Angelo Department of Biology Hamden Hall Country Day School 1108 Whitney Avenue Hamden, Connecticut 06517 Phone: 203-752-2600 Fax: 203-752-2651 e-mail:

I. INTRODUCTION Daphnia magna is a common freshwater crustacean (Olson; 2011). They are found in lakes, ponds, and streams and are about 3-5 mm in size (Russell; 2012). Because they have a transparent, colorless shell, their internal organs are visible under a microscope (Figure 1) (Clare; 2002). They also have a single compact eye (Russell; 2012). Despite their nickname, the water flea, they are related to shrimp and crabs (Clare; 2002). They are called water fleas because of the way they swim; their strong second antenna propels them forward, and they end up swimming in a jerky motion (Russell; 2012). D. magna is an important contributor to the food chain because many fish depend on them for their survival (Russell; 2012). D. magna eat small microorganisms and algae, and their legs inside the shell carapace are used to collect the food from the environment (Russell; 2012). They can also eat yeast in the laboratory (New World Encyclopedia; 2008). Even if different individuals of D. magna are compared, their appearance and color can differ depending on their environment (Clare; 2002). The life span of D. magna varies as the environment changes (Clare; 2002). The optimum temperature for D. magna is 20-25o C, and they normally live for about 40 days (Clare; 2002). D. magna shed their skin as they grow, just like other crustaceans (Clare; 2002). Their average heart rate is approximately 180 beats per minute (New World Encyclopedia; 2008). However, their heart rate can range from 91 to 521 beats per minute depending on the temperature (Corotto et al; 2010). This is because when the temperature increases, their metabolism increases, which causes the heart rate to increase so they can provide enough oxygen to their cells (Dennis; 1999). D. magna are considered an indicator species, which means they can be used to test water quality and the health of the ecosystem because they are so sensitive to environmental change (Clare; 2002). Their transparent, clear carapace allows them to be observed clearly under a microscope, and their heartbeats can be counted (Clare; 2002). They seem tolerant of being observed, and they

appear to suffer no harm when they are returned to their habitat after they have been experimented on (New World Encyclopedia; 2008). Caffeine is a naturally occurring stimulant called trimethylxanthine (Brain; 2012). A stimulant can be defined as a drug that produces a temporary increase of functional activity or efficiency of an organism (Brain; 2012). Caffeine is a drug that can become addictive, and has similar traits to cocaine and heroin (Brain; 2012). It stimulates brain function, and therefore has many other effects on the body (Brain; 2012). To a nerve cell, caffeine is metabolized like adenosine, which normally slows down activity. Instead, the caffeine blocks the receptors, so no calming effect can occur, and it causes the adrenal glands to produce adrenaline (Brain; 2012). Caffeine can also stimulate receptors in heart cells, which speed up blood, flow and increase the heart rate (Nunley; 2010). It stimulates the heart cells by blocking the enzyme phosphodiesterase, which normally works with another enzyme, cyclic AMP, to maintain the heart rate at a steady pace (Nunley; 2010). When phosphodiesterase is blocked, this system can no longer work, so the heart rate increases (Nunley; 2010). On average, caffeine increases a humanâ&#x20AC;&#x2122;s heart rate about 3 beats per minute (Nunley; 2010). Besides concentration, the change in heart rate depends on the size and tolerance of the person, because caffeine affects individuals differently (Nunley; 2010). About 90% of Americans consume caffeine every day, and about half consume more than 300 milligrams, which the U.S. Food and Drug Administration and the American Medical Association consider as the upper limit of a daily dose (Brain; 2012). One Monster energy drink contains 160 mg of caffeine, while one cup of brewed coffee averages about 115 mg (Izenburg; 2012). Athletes and students regularly rely on caffeine to maximize their performance, but there are medical dangers when too much caffeine is consumed (Over Caffeinated; 2012). When caffeine stimulates the nervous system, it can also lead to headaches and elevation in heart rate and blood pressure (Over Caffeinated; 2012). Caffeine is also a diuretic, which can make a person dehydrated (Over

Caffeinated; 2012). It makes it difficult to sleep, which is essential for the body to grow and repair itself (Over Caffeinated; 2012). Caffeine is a potentially addictive, but popular, drug that can be dangerous if it is not used in moderation. The purpose of this experiment was to see whether the heart rate of D. magna would increase, decrease, or remain the same when it was subjected to varying concentrations of caffeine. It also illustrated an approximate lethal dose of caffeine for D. magna. The lethal dose can be defined as the amount of a drug or agent that will cause death (Olson; 2011). The independent variable was the concentration of caffeine, which was measured in mg/L. The dependent variable was the number of heartbeats per minute. It is important for people to know how much caffeine is safe to consume and the effects it has on the heart, because if it is misused, there can be serious consequences. The hypothesis of this experiment was as follows: if the concentration of caffeine is altered among a set of experimental trials, then the heart rate of Daphnia magna in the higher concentrations will be greater than the heart rate of those in the lower concentrations until the concentration reaches the lethal dose because caffeine is a heart stimulant, but can also be fatal if it is not used in moderation.

Figure 1. Daphnia magna anatomy (Olson; 2011).

II. MATERIALS AND METHODS A. Materials 1. Distilled water 2. Pure white crystalline powder caffeine from Natural Food Supplements Inc. (Figures 3 and 4) 3. Spatula 4. Weighing paper 5. Electronic scale 6. Graduated cylinder 7. Daphnia magna (21) from Connecticut Valley Biological (Figure 2) 8. Jars (7) 9. Stopwatch 10. Microscope 11. Depression slides 12. Dropper pipettes 13. Cotton fibers 14. Methyl Cellulose 15. Kim wipes 16. Squeeze bottle with distilled water 17. Gloves B. Methods 1. Making the solutions Seven jars were labeled as following: Control, 800 mg/L, 400 mg/L, 200 mg/L, 100 mg/L, 50 mg/L, and 25 mg/L (Figure 5). 100 ml of distilled water was measured out,

and this acted as the control with a concentration of 0 mg/L. The purpose of the control was to determine the regular heart rate of the D. magna under the same conditions as the experimental specimens for comparison. Then, 80 mg of pure caffeine was weighed on an electronic scale using weighing paper, and this was poured into 100 ml of distilled water. The weighing process was repeated with 40 mg, 20 mg, 10 mg, 5 mg, and 2.5 mg, and each measurement of pure caffeine was poured into 100 ml of water. All the jars were lightly stirred and left over night to ensure that all the caffeine had dissolved. All of the solutions were made with the same caffeine, distilled water, electronic scale, and they were all kept at room temperature, which was about 17oC. 2. Conducting the Experiment A day before the experiment, on the day the D. magna arrived, a trial run of the experimental procedure, using distilled water, was performed, but the data was not recorded. This ensured that the data would not be altered due to experimenting with different techniques. A dropper pipette was used to transfer one D. magna to a depression slide. A Kim wipe was used to draw off excess culture water from the slide, so the concentrations of caffeine wouldnâ&#x20AC;&#x2122;t be compromised. Three drops of the control solution were added on top of the D. magna using a dropper pipette. Then, one drop of methylcellulose was added on top of the D. magna, and cotton fibers were placed around it so it was unable to move. Then, the depression slide was placed on the microscope stage. Once one minute had passed in order for the D. magna to ingest the solution, the microscope light was turned on. The number of heartbeats was counted for 15 seconds while another person held a stopwatch, and data was recorded in the lab notebook. Afterwards, the heartbeats were multiplied by 4 to determine the number of beats per minute (bpm). The heartbeats counted during the 15 seconds could have been

off by 1, and therefore the degree of uncertainty was +4 bpm. The data was quantitative, because the dependent variable was the number of heart beats. The microscope light was immediately turned off so the temperature would not increase. The D. magna was removed with a pipette, and placed into another jar filled with culture water, where all of the D. magna used in the experiment went. The depression slide was rinsed off with distilled water and dried with a Kim wipe. This whole process was repeated for each experimental solution, and then two more trials were conducted for a total of three replications to ensure accuracy. The solutions were made on 4/5, and the experiment was conducted 27 hours later. The entire experiment was done on 4/6, and it took about 5 hours. The experiment was performed during one day so the temperature, light and condition of D. magna would be constant throughout the whole procedure. Other constants were the microscope, person counting, stopwatch and the exposure time. After the experiment, the chi-square was calculated. Figure 2. Daphnia magna.

Figure 3. Caffeine.

Figure 4. Caffeine powder.

Figure 5. Solutions of caffeine.

III. RESULTS Quantitative data was taken on 4/6 with one D. magna used for each group and trial. Therefore, 21 individuals of D. magna were used in this experiment. The average heart rate ranged from 202.67 bpm in the solution of 200 mg of caffeine per liter to 104 bpm in the solution of 400 mg of caffeine per liter (Table 1). Raw data can also be found in the lab notebook, but it was recorded in the report because each D. magna was different for each trial and group, so no averages were taken until after the entire experiment was done. Table 1. Data Table for the Effect of Varying Concentrations of Caffeine on the Heart Rate of Daphnia magna.

Number of beats per 15 seconds (Raw Data) Concentration Trial 1



of Caffeine

Number of

Number of


beats per 15

beats per



Control (0

Trial 2

Trial 3






25 mg/L






50 mg/L






100 mg/L






200 mg/L






400 mg/L






800 mg/L







To determine whether the change of heart rate was significant, the chi-square test was used. The null hypothesis was as follows: if caffeine has no affect on the heart rate of D. magna, there will be no significant difference between observed results and the control value of 133.33 bpm. The chi square test was carried out using control and observed data values, the chi square equation, and a standard chi square critical values table. The degree of freedom was calculated by subtracting 1 from the number of experimental classes, making it 5. The probability used was .05. The equation for Chi Square was as follows: đ?&#x2018;&#x2039;!=

 (đ?&#x2018;&#x153;đ?&#x2018;?đ?&#x2018; đ?&#x2018;&#x2019;đ?&#x2018;&#x;đ?&#x2018;Łđ?&#x2018;&#x2019;đ?&#x2018;&#x2018;  đ?&#x2018;Łđ?&#x2018;&#x17D;đ?&#x2018;&#x2122;đ?&#x2018;˘đ?&#x2018;&#x2019;đ?&#x2018;  â&#x2C6;&#x2019; đ?&#x2018;&#x2019;đ?&#x2018;Ľđ?&#x2018;?đ?&#x2018;&#x2019;đ?&#x2018;?đ?&#x2018;Ąđ?&#x2018;&#x2019;đ?&#x2018;&#x2018;  đ?&#x2018;Łđ?&#x2018;&#x17D;đ?&#x2018;&#x2122;đ?&#x2018;˘đ?&#x2018;&#x2019;đ?&#x2018; )! expected  values  

25 mg/L= (162.67â&#x20AC;&#x201D;133.33)2 = 6.46 133.33

50 mg/L= (170.67â&#x20AC;&#x201D;133.33)2 = 10.46 133.33

100 mg/L= (185.33â&#x20AC;&#x201D;133.33)2 = 20.28 133.33

200 mg/L= (202.67â&#x20AC;&#x201D;133.33)2

= 36.06


400 mg/L= (104.00â&#x20AC;&#x201D;133.33)2 133.33

= 6.45

800 mg/L= (134.67â&#x20AC;&#x201D;133.33)2 = 0.01 133.33

X2CALC= 6.46+10.46+20.28+36.06+6.45+0.01= 79.72

X2TABLE= 11.07 (Table 2)

Table 2. Chi Square Table. (Williams; 2012)

Since the calculated chi square is greater than the chi square on the table at .05 probability, the difference between experimental trials and the controls is significant, and therefore, due to more than chance alone (Table 2).

IV. DISCUSSION The results presented here support the original hypothesis that if the concentration of caffeine is altered among a set of experimental trials, then the heart rate of Daphnia magna in the higher concentrations will be greater than the heart rate of those in the lower concentrations until the concentration reaches the lethal dose. The heart rate of D. magna increased the most in 200 mg of caffeine per liter, while the heart rate decreased the most at 400 mg/L (Graph 1). In 200 mg/L, the heart rate averaged 202.67 bpm, a 52% increase compared to 133.33 bpm for the control (Table 1). In 400 mg/L, the heart rate averaged 104 bpm (Table 1). The heart rate steadily increased from the control to 200 mg/L, but had a sudden decline at 400 mg/L, showing that it was over the lethal dose (Graph 1). It was assumed that the D. magna were dying since the heart rate was about 30 beats per minute, or 28%, lower than the control (Table 1). The heart rate increased to 134.67 beats per minute at 800 mg/L, which was about the same as the control (Graph 1). The chi square results support the hypothesis because the calculated chi square is greater than the chi square shown on the table, showing that the difference between the experimental trials and control are significant (Table 2). Therefore, caffeine does have a stimulatory effect on the heart rate of D. magna. The deviation of respected results in the solution of 800 mg of caffeine per liter could have been due to experimental error or the variation between the individuals of D. magna. Each D. magna tested were the same species, but they could have been different ages or at different health statuses. This could explain why each heart rate differed so much in each trial. Also, caffeine has a different effect on individuals. D. magna were given one minute to uptake and metabolize the caffeine in the solution, so the data recorded might not have truly reflected the effect of 800 mg of caffeine per liter if they only had ingested some of it. Most of the heart rates recorded were lower than expected, since the normal heart rate of D. magna is 180 bpm, and this was possibly due to the temperature of the room, which was 17o C (New World Encyclopedia; 2008). This was lower than the optimum

temperature for D. magna, which is 20-25oC (New World Encyclopedia; 2008). The averages show that caffeine does indeed raise the heart rate of D. magna, just as it does with humans (Nunley; 2010). However, when it reaches high concentrations, it may be lethal (Graph 1). Graph 1. The Effect of Varying Concentrations of Caffeine on the Heart Rate of Daphnia magna.

The Effect  of    Varying  Concentrations  of   Caffeine  on  the  Heart  Rate  of  Daphnia   magna   Number  of  Heart  Beats  per  Minute  

250 200,  202.7   200  

100, 185.3   50,  170.7   25,162.7   800,  134.7  

150 0,  133.3   400,  104   100  


0 0  










Concentration of  Caffeine  (mg/L)  

A few other possible experimental errors could have occurred, such as controlling the temperature and counting the heartbeats. The light was left on once, and although it was given about a minute to cool off, it still could have affected the temperature under the microscope. It was also difficult to count every heartbeat because sometimes the heart was hidden and the D. magna was jumping around. The heartbeats were counted as accurately as possible, but it was difficult to ensure the

results were exact. Also, since the beats were counted for 15 seconds, 4 when converted to beats per minute magnified any mistakes, and that is why the degree of uncertainty is +4. There are a few possible ways to improve this experiment. The original intent was to see how varying concentrations of caffeine affected the heart rate of D. magna, and this intent was not modified. It would be ideal to use only one D. magna per trial so the effect of caffeine could be seen for that individual. This would eliminate the wide variation of heart rates among individuals. However, it would be difficult to do this because the D. magna would get more and more stressed every time a new solution was tested. Also, more trials could be done to ensure that the results are accurate. The D. magna should be exposed to the solution for longer than one minute for them to fully uptake all of the caffeine and metabolize it. A follow-up experiment could be done testing the effect of exposure time of caffeine on the heart rate of D. magna. The lethal dose of caffeine could also be tested more thoroughly. In conclusion, caffeine increases the heart rate of Daphnia magna until it reaches the lethal dose, and then it decreases. D. magna reflects the effect of caffeine on humans, and therefore, caffeine should be used in moderation.

V. LITERATURE CITED 1. Author Unknown. "Daphnia." New World Encyclopedia. 28 Aug. 2008. Web. 31 Mar. 2012. <>. 2. Author Unknown. "Over Caffeinated." Caffeine., 2012. Web. 07 Apr. 2012. <>. 3. Brain, Marshall, Charles W. Bryant, and Matt Cunningham. "How Caffeine Works." HowStuffWorks. HowStuffWorks Inc., 2012. Web. 31 Mar. 2012. <>. 4. Clare, John. "Daphnia." July 2002. Web. 31 Mar. 2012. <>. 5. Corotto, Frank, Darrel Ceballos, Adam Lee, and Lindsey Vinson. "Making the Most of the Daphnia Heart Rate Lab; Optimizing the Use of Ethanol, Nicotine and Caffeine." The American Biology Teacher 72.No. 3 (2010): 176-79. Print. 6. Dennis, Bill. "Re: Why Does the Heart Rate of Daphnia Increase with Temperature?" MadSciNet: The 24-hour Exploding Laboratory. MadSci, 4 Feb. 1999. Web. 07 Apr. 2012. <>. 7. Izenburg, Neil. "TeensHealth." Caffeine. The Nemours Foundation, 2012. Web. 21 Apr. 2012. <>. 8. Nunley, Kimberly. "The Effect of Caffeine on Heart Rate." LIVESTRONG.COM. Demand Media Inc., 18 Mar. 2010. Web. 07 Apr. 2012. <>. 9. Olson, Andrew. "Caffeine and Heart Rate: A Pharmacological Study Using Daphnia Magna." Science Fair Project Ideas, Answers, & Tools. Science Buddies, 26 Oct. 2011. Web. 31 Mar. 2012. <>. 10. Russell, Eric. "The Biology Classics: Daphnia - Heart." Home Page. BioMEDIA ASSOCIATES, LLC, 2012. Web. 21 Apr. 2012. <>. 11. Williams, Jim. "Probability." Faculty Websites @ Southwest Tennessee Community College. Web. 25 Apr. 2012. <


Jack R. Dowe*

Department of Chemistry, Hamden Hall Country Day School

Yale University, New Haven, CT, USA, Department of Ecology and Evolutionary Biology*

Correspondence and reprint requests to: Kathryn Richards-Hrdlicka Department of Ecology and Evolutionary Biology Yale University Ecology and Evolutionary Biology Laboratories 165 Prospect Street New Haven, Connecticut 06511 Phone: 203-432-5259 e-mail:

Funded by Yale University and by Yale University Ecology and Evolutionary Biology Laboratory

I. ABSTRACT The fungal pathogen Batrachochytrium dendrobatidis (Bd), the causative agent of the disease chytridiomycosis, has been implicated in the worldwide decline of amphibian populations. Bd is currently the largest infectious disease threat to global biodiversity; however, very little is known about Bd in Northeastern America. Northeastern American Amphibian DNA samples, collected on epidermal swabs and preserved in 70% ethanol, have been extracted, and tested for the prevalence and zoospore load of Bd via qPCR. The following four hypotheses from a total of ~ 600 swabs: There is a lower prevalence and zoospore load (1) in amphibians from Maine than Connecticut, (2) in younger age classes (either adult or adolescent), (3) in species other than the bullfrog, and (4) in species from New England than the rest of the world. To date I have extracted 368 swabs and analyzed 276 of these for the presence of Bd. One hundred and twelve samples tested positive for the presence of Bd, and the zoospore load per individual amphibian ranged from 0.014 â&#x20AC;&#x201C; 12.076 zoospores per reaction volume, as measured by qPCR. From the data analyzed, there is 83% incidence that amphibians from Maine had a lower prevalence and zoospore load of Bd than those from Connecticut, a 60% incidence that amphibians of younger age classes had a lower prevalence and zoospore load of Bd, and a 73% incidence that amphibians other than the bull frog had lower prevalence and zoospore load of Bd. The comparative average zoospore load of infected amphibian DNA in New England was 6.912 zoospores per reaction volume, while infected amphibian DNA from the rest of the world had an average zoospore load per reaction volume of 10,000. My goal is to identify the origin of Bd via qPCR assays and genomic mapping. After the origin of the fungus has been determined, scientists can begin to study the evolutionary changes that the amphibian populations at the fungusâ&#x20AC;&#x2122;s origin have experienced, and create techniques of inhibition at the genetic level.

II. INTRODUCTION In the 1950s, Lancelot Hogben came up with a pregnancy test known as the Bufo Test, in which a womanâ&#x20AC;&#x2122;s urine was injected into a female frog, and if the frog produced eggs within 24 hours the woman was pregnant (Keynes). This technique was globalized and the special female frogs from Central Africa were sent across the globe. What scientists and pregnant women didnâ&#x20AC;&#x2122;t know was that after the urine was injected into the female frogs a fungus began to grow. This fungus was Bd, and as the frogs were sent across the world, so was the fungal pathogen. This sequence of events has established Central Africa as a possible origin for Bd; however fossils, dating back over 1,000 years have been found in the New England area that exhibit strains of Bd, so Northeastern America is also a possible origin of Bd. Chytridiomycosis is an infectious disease of amphibians, caused by the chytrid Bd, a nonhyphal zoosporic fungus (Whitaker et al.). The fungus is capable of causing sporadic deaths in some amphibian populations and 100% mortality in others. There is no effective measure for control of the disease in wild populations. The disease has been proposed as a contributing factor to a global decline in amphibian populations that apparently has affected 30% of the amphibian species of the world. Over just the past 30 years, Bd has caused the catastrophic decline or extinction (in many cases within a single year) of at least 200 species of frogs, even in pristine, remote habitats (Skerratt et al. 2007). The life cycle of Chytridiomycosis can be seen in Figure 1 below: fecycle.cfm Figure 1 - The life cycle of Chytridiomycosis. In the substrate-independent part of the life stage (see “discharge papilla forms” portion of the Figure 1), flagellated zoospores are motile (ability to move spontaneously or actively) and free living. By contrast, in the substratedependent part of the life cycle (after infecting a host), a zoospore encysts and develops into a reproductive thallus (not labeled) with a single sporangium, which produce and release new zoospores. Genetic analyses of Bd isolates have been used to attempt to differentiate between the novel and endemic pathogen hypotheses. Preliminary evidence supports the novel pathogen hypothesis, in that geographically disparate isolates appear genetically similar, and relatively little variation exists globally. However, this evidence is not definitive, because a source population with higher genetic

diversity has yet to be identiďŹ ed, and even recently developed markers have few alleles per loci, making it difďŹ cult to define spatial population structure (Rosenblum). There have been twenty-four published experimental infection studies with twenty-one amphibian species, including one salamander, two toads, and eighteen species of frogs, with the primary aim being to estimate susceptibility to infection and resulting mortality. Of ninety separate experiments, twenty have been on tadpoles, sixty-six on post-metamorphic adult amphibians, and four that included both stages. Although methods have differed substantially, some preliminary conclusions can be made. In all of these experiments 90â&#x20AC;&#x201C;100% mortality was observed after infection with Bd (Collins). There is also no cure yet for wild amphibian populations infected by Bd, though some species are much less susceptible than others. Most of the treatments tried on captive animals (increasing temperature, increasing salinity, various antifungal chemicals, the antibiotic chloramphenicol, and bio augmentation) would be impossible or impractical to carry out in the wild, so practical treatment pathways must be identified, tested in captivity, and introduced into the wild amphibian populations of the world (Whitaker et al.).

III. MATERIALS AND METHODS Amphibian DNA was collected in 5 New England states and New York (study region). The amphibian collection centers sent DNA collected on epidermal swabs and preserved in 70% ethanol, whole tadpoles, and the arms, legs and gizzards of the amphibians. The amphibian DNA samples were all held in labeled test tubes and were separated by date and location of collection. The amphibian DNA was extracted following an optimized extraction protocol, devised by my Co-mentor, Mrs. Richards-Hrdlicka, at Arizona State University. The procedure included a series of DNA purification steps, which employed the reagent Prep Man Ultra from Applied Biosystems of Forest City, California, which can be used to prepare a DNA template from bacteria, yeast, filamentous fungi, both from a plate or from tissue smears. The newly extracted amphibian DNA was then either cooled in a freezer at 0째C for later use, or immediately diluted using thirty-six microliters of ultra-purified water and four microliters of purified DNA template. The purified DNA template was then loaded into a ninety-six well Protein Chain Reaction (PCR) Plate from Bio Rad of Hercules, California , leaving the fourth, eighth, and twelfth columns empty to inhibit cross well contamination. The first three columns down to the third row of wells were used for the positive controls of this experiment, which were 100 -zoospore, 10 -zoospore and 1-zoospore samples of Bd infected amphibian DNA. The eighth row of the ninth, tenth and eleventh columns was used for the negative control (ultra-purified water). After the loading was completed, an optical seal was applied to the PCR Plate and the plate was spun down in the centrifuge before proceeding to the qPCR analysis procedures. The covered PCR plate was then placed into the qPCR machine, while the specialized assay was loaded. The DNA sample labels were entered into the virtual PCR plate and the assay was started.

Next, the purified amphibian DNA was tested for the prevalence and zoospore load of Bd via Real-Time Polymerase Chain Reaction (qPCR). Real-Time Polymerase Chain Reaction is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, Real Time-PCR enabled both recognition and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional standardizing genes. The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time. As opposed to standard PCR, where the product of the reaction is quantified at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any doublestranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target (Pabinger et al.). The latter method was used for the assays analyzed in this project. After the two-hour qPCR assay was completed, the results of the Bd qPCR assays were analyzed. The collected data was entered into a database, along with the location of amphibian capture, its sex, age class (either adult or adolescent), the species and sub species of the amphibian, and the height, weight and length of each amphibian.

IV. RESULTS The following graphs and data table summarize all of the data collected for this project. Figure 3 - An Example of an amphibian DNA sample that has tested positive for the prevalence of Bd. The 100-zoospore control is represented by the red curves, the 10-zoospore control is represented by the yellow curves, and the 1-zoospore control is represented by the green curves.

**ΔRn is an increment of fluorescent signal at each time point. The ΔRn values are plotted versus the cycle number. The horizontal red line at ΔRn 0.1 is called the threshold in this qPCR graphing system. As soon as the machine detected that the amphibian DNA samples had a zoospore load per reaction volume of ΔRn 0.1, the DNA sample exhibited a prevalence of Bd and was considered a positive sample. The qPCR instrument determines the exact zoospore load of an amphibian DNA sample to an uncertainty ±0.0001, by determining the cycle at which the amphibian DNA sample’s average zoospore load per reaction volume reached the threshold. The amphibians exhibiting the largest

zoospore load per reaction volume of Bd crossed the threshold in the fewest number of cycles because the Bd fungi did not need to be heavily replicated for it to reach ΔRn 0.1. The DNA samples that exhibited low zoospore load per reaction volume crossed the threshold after the largest amount of qPCR cycles because the Bd had to be amplified greatly before the qPCR machine was able to detect a ΔRn of 0.1. There is a gap in the graph and missing data points on the area of the plot of ΔRn versus cycle between twenty-two and twenty-four cycles. The qPCR machine does not collect data between these intervals for reasons that are not well understood. Figure 3 is an example of the graph of the positive controls: The 100-zoospore control is represented by the red curves that cross the threshold after the fewest amount of cycles (twentyeight cycles), the 10-zoospore control is represented by the yellow curves that cross the threshold after thirty-two cycles, and the 1-zoospore control is represented by the green curves that cross the threshold after the greatest amount of cycles (thirty-six cycles). The static that is seen before twenty-two cycles is the result of the machines inability to process a positive sample until the Bd infected amphibian DNA has been replicated hundreds of thousands of times. Because the zoospore load per reaction volume of the experiments controls has not reached ΔRn 0.1, the data collected fluctuates sporadically between a ΔRn of 0.01 and 0.001. For non-control wells of the qPCR runs, if either all three wells or two of the three wells of the amphibian DNA sample crossed the threshold the sample was considered positive. If only one of the three wells of the amphibian DNA sample crossed the threshold that particular sample would have to be retested for the prevalence and zoospore load of Bd. If the retested amphibian DNA sample still exhibited the same results it was not considered in the data, but a positive or negative result was considered and added to the database.

Figure 4 - An Example of an amphibian DNA sample that has tested negative for the prevalence of Bd. The ultra-purified water control is represented by the purple lines and curves.

**ΔRn is an increment of fluorescent signal at each time point. The ΔRn values are plotted versus the cycle number. Figure 4 is an example of the graph of the data for the negative control obtained in these experiments. Because the water exhibits no prevalence or zoospore load of Bd it does not cross the threshold of the ΔRn versus Cycle Plot. For non-control wells of the qPCR runs, if all three wells of the amphibian DNA sample do not cross the threshold the sample was considered negative. Of the two 276 amphibian DNA samples that were tested, 164 tested negative for the prevalence of Bd, and 112 tested positive for the prevalence of Bd. This discrepancy in the prevalence of Bd cannot be attributed to any one cause but could possibly include the location of amphibian collection, the temperature of the amphibians’ habitat, and the wind patterns in the amphibians’ habitat. Therefore, a generalization about the percentage of amphibian DNA samples

that will test positively or negatively for the prevalence of Bd cannot be determined for the reasons listed above and also because there was not a wide enough range of data collected. Table 1: Compiled data collected after completing a Z test to find the percent incidence of prevalence and zoospore load of the tested amphibian DNA Hypothesis

Percent Incidence of Prevalence and Zoospore Load

1) Lower prevalence and zoospore load in


Maine than Connecticut 2) Lower prevalence and zoospore load in


younger age classes 3) Lower prevalence and zoospore load in


species other than the bullfrog 4) Location

Average Zoospore Load (zoospores per reaction volume)

New England


Rest of World

â&#x2030;&#x2C6; 10,000

Two hundred and seventy six amphibian DNA samples have been assayed via qPCR thus far, and entered into a Z test program written using R programming software. The Z test is a statistical test for the distribution of the test statistic under the null hypothesis that can be approximated by normal distribution. The data compiled in Table 1 clearly show that the percent incidence of prevalence and zoospore load of Bd is lower in Maine than in Connecticut, in younger age classes (either adult or adolescent), and in species other than the bullfrog. The location hypothesis illustrates that amphibians in Northeastern America exhibit a vastly lower average zoospore load per reaction volume, which measures the level of infection, than amphibians in the

rest of the world. The average zoospore load per reaction volume in the rest of the world is over 100,000% larger than the average zoospore load per reaction in the study region. Limitations and Improvements of Results The limitations of the data collected include a lack of study of the ecosystems in Maine and Connecticut, which can greatly affect the prevalence and zoospore load of Bd infected amphibians. In order to obtain more significant results, a close study of wind patterns, temperature, snow and rainfall, and loss of habitat should be conducted. Because an overwhelming amount of the amphibian DNA processed at the Yale University Laboratories was from Green Frogs, the precision of the third hypothesis is questionable. The database did not contain enough DNA samples from a wide range of species to completely test the effect of Bd on varying amphibian species. Overall, the accuracy of the data collected could be easily improved by collecting a wider range of amphibian species from varying locations, as well testing more amphibian DNA samples in general.

V. DISCUSSION This project has successfully tested the effect of location, species, and age of amphibians on the prevalence and zoospore load of amphibian DNA samples collected in the study region. Amphibians in Northeastern America exhibit a lower prevalence and zoospore load if: (1) the amphibians are from Maine as opposed to Connecticut, (2) if the amphibians are of a younger age classes, and (3) in species other than the bull frog. From the data collected in this project a more effective fungal model of Bd can be created by scientists, while conservationists have a better idea of where to attack the fungus, at what age to induce treatment, and which species of amphibians to treat first. The significance of the miniscule average zoospore load per reaction volume of the Bd infected amphibian DNA collected in the study region, is that it shows that amphibians in Northeastern America have either: undergone some kind of evolutionary mutation that allows them to inhibit the excessive growth and spread of Bd, or live in an ecosystem that inhibits the excessive growth and spread of Bd. The genomes of amphibians in Northeastern America may have mutated, allowing the development of resistance to the emerging fungal pathogen Bd. The amphibians in the rest of the world exhibit almost no immunity to the fungal pathogen, so genetic analysis and comparison, as well as a comparison of the effect of varying ecosystems on the prevalence and zoospore load of Bd should be completed. In the case that a genetic mutation that inhibits the growth and spread of Bd is discovered in amphibians in Northeastern America, the roots of the disease caused by Bd can be defined and treatment plans can be devised. Induced genetic mutations have been successful in past scientific work. Dr. Jacques R. Fresco of the Princeton University Molecular Biology Department, who authored â&#x20AC;&#x153;Site-Specific Self-Catalyzed DNA Depurination, the Basis of a Spontaneous Mutagenic Mechanism of Wide Evolutionary Significanceâ&#x20AC;?, showed that after the specific gene that undergoes

mutation to either cause or inhibit a disease is found, genetic modifiers can be tested and then used to control the mutations of that certain gene (Fresco et al.). This allows for a controlled genetic mutation to occur that could very well inhibit the growth and spread of Bd. An additional pathway of continued research would be to test the chemical combinations on the skin of amphibians from the study area. Chemicals and bacteria form a slime like layer of mucus on the skin of amphibiansâ&#x20AC;&#x2122; that acts as an antimicrobial barrier and pseudo-immune system. The mucus that amphibians in Northeastern America produce could inhibit the growth and spread of Bd, and again induced genetic mutations could control the contents of slime like layer that amphibians in the rest of the world produce. Research will now hing upon the discovery genetic causes of Bd and genetic modifiers that inhibit the growth and spread of Bd. This project has embraced the pace at which Bd must be studied. The food chain, the balance of ecosystems, and the human world depend upon the composition of all species. If a species becomes extinct, the entire biodiversity of the world is at risk. It would be like removing a link that holds together the entire food chain of the world. If amphibians, or any species of organism becomes extinct the cycle of life on earth will have to quickly adapt to the change in the different biomes of the world (Kilpatrick et al). The causes of Bd must be identified and treated before conservationists and scientists have to deal with the reverberations of losing and entire species from the biomes of the world. The next step of this project will be determining the average zoospore load per reaction volume of Bd infected amphibians in Central Africa. These results can be compared to those collected in Northeastern America, and the location that exhibits the lowest average zoospore load per reaction volume will be deemed the origin of Bd. Bd infected amphibians from the newly discovered origin of Bd, will then be genomically mapped, and this will provide a Bd infected

amphibian model. This will allow mutagenic research to begin, and a genetic modifier that will inhibit the growth and spread of Bd can be discovered, tested and mass-produced.

VI. ACKNOWLEDGMENTS I would like to acknowledge the funding, help and support of Dr. Francis P. Gasparro, Mrs. Kathryn Richards-Hrdlicka, Dr. Gisella M. Caccone, Yale University Department of Ecology and Evolutionary Biology, and the Hamden Hall Science Department. VII. LITERATURE CITED 1. Collins, James P. "Amphibian Decline and Extinction: What We Know and What We Need to Learn." Diseases of Aquatic Organisms 92.2-3 (2010): 93-99. Inter Research » DAO » v92 » n2-3 » p93-99. Web. 24 May 2012. 2. Fresco, Jaques R. "Site-Specific Self-Catalyzed DNA Depurination, the Basis of a Spontaneous Mutagenic Mechanism of Wide Evolutionary Significance. “Evolutionary Biology: Concepts, Biodiversity, Macroevolution, and Genome Evolution. By Pierre Pontarotti. Heidelberg: Springer, 2011. 3-19. Spinger for Research and Development. Springer, Jan. 2011. Web. <>. 3. Keynes, Milo. "Lancelot Hogben, FRS (1895-1975)." Notes and Records of the Royal Society 53 (1999): 361-69. Lancelot Hogben. Galton Institute. Web. 24 May 2012. 4. Kilpatrick, Marm A. "The Ecology and Impact of Chytridiomycosis: An Emerging Disease of Amphibians." Trends in Ecology and Evolution 30.10 (2009). Trends in Ecology and Evolution. Cell Press. Web. 5. Pabinger, Stephen G. "BMC Bioinformatics | Full Text | QPCR: Application for Real-time PCR Data Management and Analysis." BMC Bioformatics 10.268 (2009). BMC Bioinformatics | Full Text | QPCR: Application for Real-time PCR Data Management and Analysis. Web. 24 May 2012.

6. Rosenblum, Erica B. "A Molecular Perspective: Biology of the Emerging Pathogen Batrachochytrium Dendrobatidis." Diseases of Aquatic Organisms 91 (2009). National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 24 May 2012. 7. Skerratt, L. F., Berger, L., Speare, R., Cashins, S., McDonald, K. R., Phillott, A. D., Hines, H. B., and Kenyon, N. 2007. Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth 4: 125-134. 8. Whitaker, Kellie, and Vance Vredenburg. "An Overview on Chytridiomycosis. â&#x20AC;&#x153;Chytridiomycosis. University of California, Berkley, 1 June 2011. Web. 03 June 2012. <>.


Alexandru D. Buhimschi *


Department of Chemistry

Hamden Hall Country Day School, Hamden, Connecticut, USA.

Correspondence and reprint requests to: Francis P. Gasparro, Ph.D. Department of Chemistry Hamden Hall Country Day School 1108 Whitney Avenue Hamden, Connecticut 06517 Phone: 203-752-2600 Fax: 203-752-2651 e-mail:

Funded by: Immunolight, LLC Duke University Department of Biomedical Engineering, Chemistry, and the Fitzpatrick Institute for Photonics

I. ABSTRACT Psoralens are used for treatment of hyperproliferative skin conditions due to an ability to form DNA adducts upon excitation with UV photons. Such adduct formation leads to impaired DNA replication and cell death. Although UVB and psoralen have been used clinically, the extent and nature of adduct formation has yet to be reported. Interest in psoralen intracorporeal photochemotherapy resulted from development of energy sources and synthetic tags capable of insitu photon delivery. Functional optimization of such tagged psoralens depends upon their ability to intercalate, UV wavelength, and the technique for adduct detection. By convention, UVA is used due to an extinction coefficient (2,016 cm-1M-1) sufficient to facilitate adduct formation without psoralen degradation. However, psoralens have an even greater extinction coefficient (11,800 cm1

M-1) in the UVB region, which may be more efficient when carrying out tumor-targeted delivery of

psoralens. Due to the structural nature of psoralens that allows energy absorption and the surface of the arrays, SELDI-TOF, a proteomic technique that combines chromatography with mass spectrometry, may prove more efficient than conventional detection techniques. Our group optimized a new rapid and quantitative adduct detection protocol using SELDI-TOF. An alternating A-T oligonucleotide (3,025m/z) was synthesized for 4′-aminomethyltrioxsalen (AMT) adduct formation. DNA and AMT solutions were irradiated with UVB (300 nm) for 12 min. Since DNA is negatively charged, a quaternary ammonium-coated surface (Q10 ProteinChip array) was utilized for sample binding. Using our optimized matrix protocol consisting of 3-hydroxypicolinic acid and overlaid with ammonium citrate, an adduct peak (3282m/z), consistent with UVB dose dependence, was obtained. Adduct formation was detected as early as 1.5 min. Due to degradation or adduct site depletion, adduct formation began to plateau at 9-12 min. The rate of adduct formation was faster than UVA. This new rate confirms the possibility of using UVB in a pseudo “flash”-photolysis photochemotherapy setting. The higher energy of UVB and the rapid adduct formation may prove useful in the field of developing synthetic psoralens. In addition, SELDI-TOF has proven effective for detection of photoadducts. Our results demonstrate new modalities for assessing and activating synthetic psoralens as anti-cancer drugs.

II. INTRODUCTION Mechanism: Psoralen, a furocoumarin, has long been studied for its medicinal properties. However, recent advances in the past century allow for a greater understanding of psoralen’s treatment mechanism. Psoralen molecules are planar and able to migrate easily between DNA base pairs (intercalation), particularly adenine and thymine sites. Psoralen derivatives possess varying degrees of affinity for nucleic acids and are therefore more or less effective drugs. This process of intercalation positions the psoralen in the optimal orientation to form photoadducts. The planar molecular structure and bonding arrangement also makes it effective at absorbing energy. The key area of excitation for psoralen derivatives is in the ultraviolet range. An absorbance spectrum of 4’aminomethyltrioxsalen (AMT) shows a predominant peak at 303nm (See Figure 1). Photoexcitation of an intercalated psoralen leads to the photoactivation of either the furan or pyrone double bond1. Upon excitation, psoralens can degrade, generate singlet oxygen, or form photoadducts (See Figure 2). It is believed that adduct formation plays a crucial role in psoralen’s therapeutic effects. Adducts, once formed in the nucleic acid sequence, are difficult to repair. The absorbance spectra of crosslinks and monoadducts are markedly different from the free molecule. Thus, the photoreversal of adducts, while characterized, occurs to a lesser extent2. The enzymes responsible for DNA replication cannot bypass these adducts and cell division and density is correspondingly slowed. This Figure 1. Absorbance spectrum of 4’aminomethyltrioxsalen (AMT) showing the shoulder typical of psoralens in the UVA range.

property makes psoralens very effective at combating uncontrolled and possibly

malignant cell division. However, unlike other modifiers of DNA, psoralens can be triggered selectively with UV light, making them very therapeutically applicable. Treatments and Applications: In modern times, psoralens went from ancient remedies to medical tools. In 1947, Fahmy isolated psoralens from plants. In addition, the synthesis of 8-methoxypsoralen (8-MOP) occurred in Germany in the 1930s. In 1948, El-Mofty completed the first clinical reports of vitiligo treatment with psoralen. In 1974, Parrish applied psoralen therapy to the treatment of psoriasis when he applied the drug to the affected area and Figure 2. (A) 8-methoxypsoralen (8-MOP) crosslink. (B) AMT crosslink.

subsequently exposed the patient to UVA irradiation. In 1985, Gasparro developed the photopheresis technique to

treat cutaneous t-cell lymphoma3. The variety of applications made psoralen phototherapy a very active field of study. However, all such applications rested on the irradiation of superficial tissues. In the case of photopheresis, psoralen-treated blood is removed from the patient, cycled through an irradiation apparatus and afterwards reinfused4. In spite of the numerous advances, questions still exist as to whether psoralens could ever be used to treat conditions inside the body and in areas impenetrable to UV irradiation. About 1,638,910 new cancer cases are expected to be diagnosed this year in the United States. The traditional approaches of chemotherapy, surgery, radiation therapy, photodynamic therapy with ROS-generating drugs, photopheresis have been used successfully to treat various cancers. However, the absence of a truly targeted therapeutic approach that accounts for the innate variability in cancer conditions has made treatments less effective and side effects increasingly more difficult to cope with.

Scaffidi et al. has described the synthesis of a novel photonics agent (nanoscintillator) made with metal nanoparticles (Y2O3) that can convert high energy X-rays to UVA for use in activating a psoralen drug. A psoralen, once attached to this downconverting nanoscintillator, can be delivered to a tumor and activated with a photon generated in-situ. By tagging a psoralen to a fragment of the HIV-1 TAT cell penetrating/nuclear targeting peptide, the drug can be introduced directly to the DNA sequence where it can begin adduct formation. Experiments performed have also shown that in the absence of X-ray irradiation, these drugs have limited toxic effects5. Such studies have been the first demonstration of cell death or growth inhibition by X-ray excited, psoralen-activating nanoscintillators. The nanoscintillators that activate photochemical drugs are the treatment tools of future therapies. Moreover, the X-ray radiation doses could be reduced to a point where fatigue and nausea become minimal. This new approach is both more targeted and noninvasive than previous attempts to cure cancers. Photopheresis and psoralen-functionalized nanoscintillators both use radiation in the UVA range. Numerous reports use a 365 nm wavelength to activate psoralens due to the sufficient absorbance to generate adducts while subsequently not causing degradation. To this date, no articles have made a significant investigation into the extent and characterization of adducts formed in other ranges of the UV spectrum. Our group, upon examination of the absorbance spectra of AMT and 8MOP decided to investigate the application of UVB (300 nm) light. The greater extinction coefficient in UVB (11,800 M-1cm-1) compared to UVA (2,016 M-1cm-1) should facilitate adduct formation at a faster rate than UVA. While in the short-term, adduct formation for UVB should be greater, long-term adduct levels generated with UVB and UVA should approach one another. Moreover, the shorter irradiation times could be important in limiting toxic effects caused by exposure to X-ray radiation.

Adduct Detection: Numerous methods exist to detect psoralen adducts. An enzyme protocol is applied to the irradiated samples to digest the DNA down to nucleosides6. Such analysis allows for the differentiation between monoadducts and crosslinks. In the past, our group optimized such a protocol by substituting the very active Benzonase for Nuclease P17. However, our group was solely concerned with the quantitation of adduct levels across UVA and UVB experiments, making the type of adduct irrelevant for our objective. While matrix assisted laser desorption ionization (MALDI) mass spectrometry has been used, we report the first ever analysis of psoralen-modified oligonucleotides with surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF). SELDI-TOF, a technique used mostly in proteomics, combines the binding capabilities of chromatography with the detection of mass spectrometry. MALDI-TOF, which is solely matrix assisted, involves spotting and drying according to either thin-layer or dry-droplet techniques. SELDI has various chips, each with different affinities for analytes displaying a certain characteristic (e.g. hydrophobicity, metal affinity, positively charged, etc.). Oligonucleotides are negatively charged and should bind to a chip with a positive surface. Using our new protocol, our group has shown the quantitative nature of SELDI analysis and the high level of sensitivity and specificity generated by the mass spectra. Significance: We report a novel method of generating and detecting psoralen photoadducts. In particular, our results may prove significant in the nanoscintillator mediated treatment studies of Scaffidi et al. The rapidly developing field of nanomedicine and the growing attempts to craft a targeted approach towards cancer therapies may leverage the unexploited potential of psoralens as photochemotherapeutic agents. The activation of psoralens relies upon the means of irradiation. Our models and results indicate a significant advance over the current means of generating adducts, particularly those intended to mediate the hyperproliferative condition of cancer.

III. MATERIALS AND METHODS Reagents: All drugs were purchased from Sigma, St. Louis, MO and were ultrapure grade. A ten base pair alternating adenine and thymine oligonucleotide (AT-10) was synthesized at the Yale University Oligonucleotide Synthesis Resource, New Haven, CT. Preparation of Irradiation Solutions: AMT’s water solubility is >104 µg/mL. A stock solution was made in water and concentration was verified spectrophotometrically with the molar extinction coefficient at 298 nm (10,000 M-1cm-1). In final irradiation solution, [DNA] was 0.5 mg/mL and [AMT] was 0.05 mg/mL8. Irradiation of Solutions: The melting temperature of AT-10 was found to be ~18ºC. To prevent helix unwinding, solutions were chilled and irradiated on ice. The solution vessel was sealed and irradiated using a panel of UVA (365 nm) or UVB (300 nm) bulbs (Model UV-1 irradiator, The Southern New England Ultraviolet Company, Branford, CT). Aliquots were taken at regular time intervals over the course of 120min. During irradiation with UVA, a UVB filter was used to shield against unwanted radiation. Prior to SELDI analysis, the time points were stored in sterile, light sensitive tubes to prevent any further activation of the AMT. Mass Spectrometric Analysis: A SELDI-TOF instrument model PBS IIC (Ciphergen Biosystems Inc., Fremont, CA) was used for all mass spectrometric analyses. Q10 ProteinChips were purchased from Bio-Rad Laboratories (Hercules, CA). Sample Binding: At first, it was hypothesized that a CM10 anionic chip would be sufficient to bind the positively charged AMT adducts. However, at the pH of our solutions, it was determined that the protonation of AMT would not be sufficient. An IMAC30 metal affinity chip bound with gallium (used to detect phosphorylated proteins in proteomic analysis) was investigated but proved too cumbersome and lengthy. Next, we proposed that a positively charged chip surface could bind

the negative DNA phosphate backbone. A quaternary ammonium chip (Q10) was used in this analysis9. During the time when our group used poly dA-dT, we noticed a significant amount of peak suppression likely caused by the enzymes remaining from the digestion protocol. DNA purification is therefore essential for solutions containing such enzymes. As a matrix, we used 40 mg/mL hydroxypicolinic acid (HPA), which is used in MALDI-TOF oligonucleotide applications. It was noted that a 0.3M ammonium citrate dibasic solution proved effective in limiting matrix saturation. The chips were preconditioned with 100mM Tris-HCl (pH 8.00). Samples were diluted in a 1:1 v/v ratio of 100mM Tris-HCl (pH 8.00) buffer. 6ÂľL aliquots of sample were placed on each chip and incubated in a humidity chamber for 1 hour. The spots were aspirated and washed equally with buffer and allowed to dry for 10 min. 1.5ÂľL HPA were placed on each spot and allowed to dry in a steady stream of air. An equal volume of ammonium citrate was added onto the chip and dried (See Figure 3) Spectral Analysis: Variability among the chip surfaces made direct comparison of different spots incorrect. As was expected, our spectra provide us with a peak for free Figure 3. An illustration of SELDI-TOF chip preparation for detection of a psoralen-modified oligonucleotide.

AT-10 and another for the AMT-modified DNA. Therefore, we decided to apply an internal standard by determining the ratio of

the TOF area between free and modified oligonucleotide. Such analysis provided us with an accurate means of taking into account spot variability. The focus of further experimentation will be the minimization of spot variability caused by the layers of DNA, HPA, and ammonium citrate.

IV. RESULTS SELDI-TOF Analysis and UV Dose Dependence: Our SELDI-TOF protocol has been shown to provide consistent spectra that indicate adduct formation according to UV irradiation time. These results suggest that SELDI-TOF, a mass spectrometer used solely for proteins, can be applied to other analytes with great success. We obtained 4â&#x20AC;&#x2122;aminomethyltriosxalen hydrochloride. Thus, in all spectra the dissociation of the hydrochloride group was taken into account when determining adducts. Figure 4 shows the adduct concentration increasing with time and the consistency with which SELDI-TOF provided results. Figure 5 illustrates the UV dose dependence of our adduct formation over the 2hr. irradiation period.

Figure 5. Line graph on log scale showing UVB dose dependence. Note the initial sharp increase followed by a plateau. Effect of UVB Irradiation on Adduct Formation: As was initially hypothesized, UVB adduct formation proved much greater than UVA in the initial 12min irradiation time. However, as irradiation time approached 2hr. adduct levels began to equal one another. With a higher extinction coefficient, the absorption of energy should be greater and promote the formation of adducts at a faster rate. However, the higher energy of UVB could also promote the photoreversal of adducts

(See Figure 6). As is seen in Figure 6, the crosslink absorbance is much greater at 300 nm than at 365 nm. Further experimentation will address longer irradiation times and effects on adduct levels. While it was originally thought that UV wavelengths less than 320nm cause only the photoreversal of previously formed adducts and degradation of non-intercalated molecules, it has now been shown that UVB doses lead to photoadduct formation. Figure 7 shows the experiment illustrating the faster rate of UVB adduct formation. Again, UVB clearly produces more AMT adducts in shorter time intervals. Total AMT adduct levels approach one another at longer irradiation times. This result suggests that using AMT in a pseudo â&#x20AC;&#x153;flashphotolysisâ&#x20AC;? setting with short irradiation times may prove therapeutically applicable with Figure 6. Absorbance spectrum of crosslink (From Olack GA et al. Photochem Photobiol 1993; 57:941-949, with permission.

downconverting nanoscintillators. A UVB producing nanoscintillator could form adducts with

Ratio TOF area (intact oligo/1st adduct)

a significantly lower X-ray irradiation time. Our results confirm that SELDI-TOF MS can




be used to generate consistent and specific


mass spectra for psoralen-modified


oligonucleotides. Lastly, our novel means


of psoralen activation may prove useful in


3 min

12 min

90 min

120 min

Figure 7. UVA and UVB adduct levels over 2hr. time course irradiation. At 3min. and 12min. UVB AMT adduct levels are greater than those produced by UVA.

the nanomedicine field of developing synthetic psoralens and nanoscintillators to combat cancers.

Classic mass spectrometric techniques involve analyzing oligonucleotides in a positive-ion mode. Our group has shown that our AT-10 oligonucleotide can be desorbed in a negative-ion mode at a laser intensity of 220. Figure 8 shows a SELDI mass spectrum with the characteristic free DNA (3025 m/z) and adducted DNA (3282 m/z) peaks. Note that the strands of ten base pair oligonucleotide are antiparallel and will anneal on others. However, in our instrument, the strands appear to separate, likely a result of the high laser intensity. A protocol was found that demonstrated a method for keeping DNA intact in MALDI-TOF by using 6-aza-2-thiothymine (ATT)10. Our group replicated this procedure but was only successful following drying of the matrix at lower temperatures to ensure that the oligonucleotide cocrystallized in intact form. Figure 9 is a mass spectrum obtained with ATT showing the double stranded oligonucleotide being detected. This has been the first ever demonstration of intact oligonucleotide desorption in SELDI-TOF MS.

Figure 8. SELDI-TOF MS Spectra. (A) Control sample of AT-10 (3025 m/z). (B) 90min. UVB irradiation dose spectrum. Note that the difference between each of the peaks is the mass of one AMT.

Figure 9. (A) Control Sample of AT-10. (B) Close-up view of intact AT-10. Note the predominant dimer peak at 6025 m/z. (C) 120 min. irradiated sample. (D) Close-up view of irradiated sampleâ&#x20AC;&#x2122;s dimer peaks. Note the peaks past 6025 m/z. These indicate AMT adduct formation. However, the actual identity of the adducts cannot be determined.

V. DISCUSSION Significance: Annual cancer diagnoses worldwide number greater than ten million. Current treatment mechanisms do not provide sufficiently targeted therapy to the inherently complex and variable condition of cancer. A theranostic approach is therefore required. Work performed by Immunolight LLC and by Scaffidi et al. provides a methodology for harnessing the potential of psoralen related compounds for combating and substantially reducing malignant cell division. Traditional approaches to psoralen photoactivation have been solely concerned with the UVA spectrum. While UVA does facilitate adduct formation, UVB could hold the potential to generate a significantly greater amount of adducts in a substantially reduced time. Psoralen activation, in particular that concerned with in vivo therapeutic application, is a balance between the extent of UV absorbance and the duration of adduct generation. Few resources have offered any elucidation on the wavelength dependence of adduct formation. Comprehensive action spectra for DNA adduct formation are required if one desires to determine the optimal activation wavelength. In the end, the “forgotten field” of psoralens may hold the potential to unlock a more targeted and therapeutically applicable approach towards curing cancer; an approach that chemotherapy, PDT, and all other methods have not been able to achieve. Further Experimentation: Psoralens are often categorized based upon their binding constants. AMT has one of the highest binding constants of all psoralens (KDNA= 150,000 M-1), making it very effective at generation adducts. However, such a high binding constant may make the method of activation less important. Our group has therefore conducted experiments with 8-MOP (KDNA= 714 M-1). It is proposed that with a psoralen of far lesser affinity, the difference between adducts formed with UVA and UVB should be far greater. Experiments have been conducted with 4’hydroxymethylpsoralen (HMT) and 8-MOP in the past, but have not been successful in generating substantial adducts due to the high ethanol concentration. Ethanol, which unwinds the

small (ten-mer) DNA helix, does not allow for the psoralens to orient themselves in proper position for adduct formation. However, the low solubilities in water of 8-MOP (36µg/mL) and HMT (41µg/mL) have made experimentation difficult. Current studies are focused on using dimethylsulfoxide (DMSO) as the proper solvent for 8-MOP and HMT solutions. Since stock solutions can be made much more concentrated, the amount of DMSO can be minimal. While DMSO also causes a lowering of DNA melting temperature, its low concentration in our irradiation solutions does not impact helix formation. Further SELDI-TOF MS protocol optimizations are required to reduce variability among chip surfaces. Our group wishes to investigate the possibility of using α-Cyano-4-hydroxycinnamic acid (CHCA) in desorbing our oligonucleotides. In addition, further tests regarding sample concentration, buffer pH, ammonium citrate concentration, and drying time are required. Insofar as proof of principle, this study is the first illustration of the ability of SELDI-TOF to detect a UVB induced psoralen-modified oligonucleotide.

VI. Acknowledgements I would like to acknowledge the help and support of Dr. Francis P. Gasparro, Immunolight LLC, and The Duke University Department of Biomedical Engineering. VII. LITERATURE CITED 1 Extracorporeal Photochemotherapy: Clinical Aspects and the Molecular Basis for Efficacy, Francis P. Gasparro, 1994   2  Wavelength dependence for the photoreactions of DNA-psoralen monoadducts. 1. Photoreversal of monoadducts, Yun Bo Shi and John E. Hearst Biochemistry 1987 26 (13), 3786-3792   3  Psoralen DNA Photobiology Volume I, Francis P. Gasparro, 1988   4  Phototherapy and Photopharmacology. The Yale journal of biology and medicine (1985) Volume: 58, Issue: 6, Publisher: Yale Journal of Biology and Medicine, Pages: 519-534   5  Activity of psoralen-functionalized nanoscintillators against cancer cells upon X-ray excitation. ACS Nano. 2011 Jun 28;5(6):4679-87. Epub 2011 May 12.   6  Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry P.F. Crain, Methods Enzymol. 193 (1990) 782-790   7  DNA Digestion to deoxyribonucleoside: A simplified one-step procedure, Quinlivan, Gregory, Analytical Biochemistry 373 (2006) 383-385   8  Monoclonal antibodies to DNA modified by 8-methoxypsoralen and ultraviolet A light, R.M. Santella, N. Dharmaraja, F.P. Gasparro, R.L. Edelson, Nucl. Acids Res. (1985) 13 (7):2533-2544.   9  An optimised sample preparation for the MALDI-ToF-MS of oligonucleotides, R. Limberg, R. Römling, M. Resch, Application MALDI Shimadzu   10  6-Aza-2-thiothymine: a matrix for MALDI spectra of oligonucleotides, P. Lecchi, H.M. Le, L.K. Pannell, Nucleic Acids Res. 1995 April 11; 23(7): 1276–1277.

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