American Society for Photobiology

ASP Conference 2016: 21-26 May 2016
Tampa Marriott Waterside Hotel & Marina


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10 - Offered Papers

Florida 2   10:00 - 12:00

Chair(s): William Ward, Kalyan Karumanchi
 
10-1   10:00  Low Level Laser Therapy and Memory Enhancement: Effect of Different Energy Doses RP Pandey*, none

Abstract: The amount of information that can be stored in short-term memory can vary. An often cited figure is 7±2 items, based on the results of a George A Millers experiment on short-term memory, published in 1956. This clinical study aimed to see the effect of different energy doses of Low Level Laser Therapy (LLLT) on memorizing capacity of the young population aged 20years to 30 years. 106 subjects with minimum of a 10th grade education with similar cultural background volunteered in the study. The subjects were divided into in Group A and Group B according to the laser set they used. The Group A consisted of 56 (Mean±SD 23.7±4.8) subjects and Group B consisted of 50 subjects (Mean±SD 24.89±3.61). Two sets of different multi-diode lasers (Q10 & Q1000) with different energy output but same wavelengths (650, 808) and energy density (38.89mJscm2) were applied for 3 minutes on the frontal and occipital region of the head of the participants simultaneously. For the purpose two equivalent tests of non-associated meaningful words were prepared. One test was used as pre test to determine the baseline working memory at start of the study. The lasers were applied twice a week for 4 weeks. Q10 with energy output of 14mJ/s was applied by group A and Q1000 with energy output of 42mJ/s was applied by group B. Depending on the type of laser applied the participants were divided into two groups. At the end on the 8th application on day 30 the participants took another equivalent "non-associated memory test" and the results were analyzed. For Group A the mean ± SD value score of pre-test was 1.7±1.7 and post test was 1.7±1.7, (p-value0.6090). For Group B the mean value score of pre-test was 2.14±1.5, and the post test score was 2.22±1.04 (p-value0.0553). Using the score of 7 as 100% memory retention, the average percentage of the data was calculated. For group A pre test scored 23.9% and post test scored 53.6% while for group B the pretest scored 30.6% and post test scored32.6%.

10-1   10:00  Low Level Laser Therapy and Memory Enhancement: Effect of Different Energy Doses RP Pandey*, none

Abstract: The amount of information that can be stored in short-term memory can vary. An often cited figure is 7±2 items, based on the results of a George A Millers experiment on short-term memory, published in 1956. This clinical study aimed to see the effect of different energy doses of Low Level Laser Therapy (LLLT) on memorizing capacity of the young population aged 20years to 30 years. 106 subjects with minimum of a 10th grade education with similar cultural background volunteered in the study. The subjects were divided into in Group A and Group B according to the laser set they used. The Group A consisted of 56 (Mean±SD 23.7±4.8) subjects and Group B consisted of 50 subjects (Mean±SD 24.89±3.61). Two sets of different multi-diode lasers (Q10 & Q1000) with different energy output but same wavelengths (650, 808) and energy density (38.89mJscm2) were applied for 3 minutes on the frontal and occipital region of the head of the participants simultaneously. For the purpose two equivalent tests of non-associated meaningful words were prepared. One test was used as pre test to determine the baseline working memory at start of the study. The lasers were applied twice a week for 4 weeks. Q10 with energy output of 14mJ/s was applied by group A and Q1000 with energy output of 42mJ/s was applied by group B. Depending on the type of laser applied the participants were divided into two groups. At the end on the 8th application on day 30 the participants took another equivalent "non-associated memory test" and the results were analyzed. For Group A the mean ± SD value score of pre-test was 1.7±1.7 and post test was 1.7±1.7, (p-value0.6090). For Group B the mean value score of pre-test was 2.14±1.5, and the post test score was 2.22±1.04 (p-value0.0553). Using the score of 7 as 100% memory retention, the average percentage of the data was calculated. For group A pre test scored 23.9% and post test scored 53.6% while for group B the pretest scored 30.6% and post test scored32.6%.

10-2   10:20  Persistence Of DNA Damage Induced By Chronic UVB Irradiation In The Human Genome R Bérubé*, University Laval and Centre Hospitalier Universitaire de Quebec Research Center ; MC Drigeard Desgarnier, University Laval and Centre Hospitalier Universitaire de Quebec Research Center; PJ Rochette, University Laval and Centre Hospitalier Universitaire de Quebec Research Center

Abstract: Exposure to UVB rays is a major risk factor in skin cancer initiation. In fact, UVB wavelengths are responsible for the formation of cyclobutane pyrimidine dimers (CPD), a pre-mutagenic damage, that lead to the C → T and CC → TT mutations found in non-melanocytic skin cancer. Recently, we have shown that a chronic irradiation with low dose of UVB (CLUV) leads to the formation of CPD that remains unrepaired (residual CPD). We then aim to determine the distribution, the localization and the impact of those residual CPD on the human genome. Four different cultures of human diploid fibroblasts were irradiated using a precise CLUV. Metaphase spreads were prepared from the irradiated cells and CPD were revealed. Chromosomes were counted and classified according to the number of sister chromatids per chromosome containing CPD (0, 1 or 2). We observed that residual CPD are tolerated in the genome and are diluted through cellular division. To localize residual CPD in the genome, we have optimized a chromatin immunoprecipitation (ChIP) protocol. Two fractions were obtained using an anti-histone 3 acetyl lysine 9 for euchromatin and anti-histone 3 tri methyl lysine 9 for heterochromatin. The amount of residual CPD induced by the CLUV according to the chromatin compaction status will be quantified using an anti-CPD ELISA. Using BrdU incorporation, we have quantified the occurrence of sister chromatids exchange (SCE) in CLUV and unirradiated cells. We have shown that the CLUV treatment catalyzes SCE as we observe 10% more SCE in irradiated when compared to unirradiated cells. This clearly indicates that residual CPD lead to genomic instability. Taken together, our results have demonstrated that exposure to chronic irradiation of UVB wavelengths leads to genomic instability through the formation of residual CPD in the genome that are not repaired but rather diluted with cell division.

10-4   11:00  Purifying GFP By Modified Three-Phase Partitioning WW Ward*, Rutgers University ; C Turner, Rutgers University

Abstract: Three-phase partitioning (TPP) has been used by our group and others to purify green-fluorescent protein. TPP has reduced processing time from several months to one or two days. In purifying recombinant GFP from transformed E. coli cells, we have made three very useful modifications. We have switched from using t-butanol to the much less expensive isopropanol and we apply TPP directly to freshly harvested, un-lysed cells. Under the influence of 1.6 M ammonium sulfate, the alcohol behaves as a low polarity solvent, dissolving the cell membrane. The mixed solvent enters through the large cell wall pores and precipitates all DNA and most protein. The aggregated macromolecules become entombed within the cells while the more soluble GFP easily exits--usually 40% pure. We employ three precipitation stages rather than one or two. Then, one round of hydrophobic interaction chromatography increases purity to 90% or higher.

10-5   11:20  The Effect of Psoralen Ring Substituents and DNA Base Composition on Photoreactivity A Trehan, Hamden Hall Country Day School ; C Wang, Hamden Hall Country Day School; M Crockett, Hamden Hall Country Day School; A Buhimschi, Yale University; H Jing, Nationwide Childrens Hospital; I Buhimschi, Nationwide Childrens Hospital; D Gooden, Duke University; F Gasparro*, Hamden Hall Country Day School

Abstract: From a library of 76 new psoralen derivatives, 9 compounds were selected based on their potential photoreactivity with DNA. Among the substituents were halogens, hydrophobic alkyl chains and aminomethyl alkyl chains. These new compounds were compared to two commonly available psoralen derivatives: 8-methoxypsoralen (8-MOP) and 4'-aminomethyl-trimethylpsoralen (AMT). The psoralens were irradiated with either UVA or UVB radiation in solutions with a series of synthetic oligonucleotides. Initial studies were performed in a self-complementary ten base alternating oligonucleotide (AT-10: A-T-A-T-A-T-A-T-A-T, Tm=20C). To test the effect of oligonucleotide stability on psoralen photochemistry, two additional self-complementary oligonucleotides were also studied ( ATGC1: G-T-A-T-A-T-A-T-A-C, Tm=24C, and ATGC2: G-C-A-T-A-T-A-T-G-C, Tm=28C). After UV exposure (at 4 C) the samples were analyzed by Matrix-Assisted Laser-Desorption-Ionization-Time-of-Flight-Mass-Spectrometry (MALDI-TOF-MS).In the 8-MOP/AMT solutions with AT-10, the original oligonucleotide was detected at 3023 Da. The 8-MOP/AMT adducted AT-10 oligos were detected at 3239 and 3280 Da respectively. These studies confirmed the well-characterized photoreactivity of AMT as compared to 8-MOP (38.5% vs 6.37%). Among the new compounds, halogen-substituted compounds were virtually unreactive in terms of adduct formation. When a 3-aminopropoxy substituent was added at the 8-position of psoralen, the level of adduct formation reached 57.5%. When a more bulky 4-methylpiperazino group was substituted, reactivity fell to 18.6%. Adduct formation was comparable when either UVA or UVB was employed. In ATGC1 and ATGC2 there were dramatic increases in AMT photoaddition (37% and 19% compared to 15% AMT in AT-10 after just a 5 min UVA exposure). All of the new psoralen compounds had been shown to be highly active in a clonogenic assay suggesting that routes other DNA modification may play a role in their efficacy. Synthesis of new psoralen derivatives with superior photoreactivity is an important goal in cancer therapeutics.

10-6   11:40  DNA and RNA Analogs for Topical Photodynamic Therapy M Pollum*, Case Western Reserve University ; L Guan, Case Western Reserve University; S Ahsanuddin, Case Western Reserve University; E Baron, Case Western Reserve University; M Lam, Case Western Reserve University; CE Crespo-Hernandez, Case Western Reserve University

Abstract: Photodynamic therapy (PDT) is an effective treatment option for a variety of skin cancers and diseases. Recently, the DNA analog 4-thiothymidine (4tT) has demonstrated efficacy as a PDT agent against rapidly-dividing malignant cells while leaving normal cells unharmed. Its phototoxic activity and low off-target effects make this thiobase a highly promising PDT candidate. However, the depth of tissues able to be treated with 4tT is limited by its absorption cutoff at ~365 nm. A thiobase photosensitizer able to absorb longer wavelengths of light is needed because longer wavelengths can reach more invasive skin cancers and diseases. Recently, we found that doubling the sulfur substitution of the nucleobase increases its photoreactivity and simultaneously shifts its absorption spectrum into the near-visible region (~395 nm) where light can penetrate more than 100% deeper into tissues. In vitro screening experiments performed in our laboratory using epidermoid carcinoma cells (A431) have revealed several doubly-substituted thiobase derivatives that are effective photosensitizers. Cell proliferation assays were used to determine the optimal thiobase doses, which do not significantly impact cell survival in the absence of UVA light (360-400 nm). Exposure of the thiobase-treated cells to a low dose of UVA light (<5 J/cm^2) results in up to 60% cell death, while the same UVA exposure has no effect on untreated cells. The demonstrated photodynamic efficacy of these novel thiobase derivatives against skin cancer cells, coupled with their expected phototoxicity in deeper human tissues than the recently reported 4tT photosensitizer, fuels the prospect of using these sulfur-substituted DNA and RNA analogs for topical PDT. [The authors acknowledge the CAREER program of the National Science Foundation (Grant No. CHE-1255084) for financial support.]



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