Structure and photophysics of indigoids for singlet fission: Cibalackrot

We report an investigation of structure and photophysics of thin layers of cibalackrot, a sturdy dye derived from indigo by double annulation at the central double bond. Evaporated layers contain up to three phases, two crystalline and one amorphous. Relative amounts of all three have been determined by a combination of X-ray diffraction and FT-IR reflectance spectroscopy. Initially, excited singlet state rapidly produces a high yield of a transient intermediate whose spectral properties are compatible with charge-transfer nature. This intermediate more slowly converts to a significant yield of triplet, which, however, does not exceed 100% and may well be produced by intersystem crossing rather than singlet fission. The yields were determined by transient absorption spectroscopy and corrected for effects of partial sample alignment by a simple generally applicable procedure. Formation of excimers was also observed. In order to obtain guidance for improving molecular packing by a minor structural modification, calculations by a simplified frontier orbital method were used to find all local maxima of singlet fission rate as a function of geometry of a molecular pair. The method was tested at 48 maxima by comparison with the ab initio Frenkel-Davydov exciton model. Published under license by AIP Publishing

Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

Impact of Nox5 polymorphisms on basal and stimulus-dependent ROS generation.

Nox5 is an EF-hand containing, calcium-dependent isoform of the NADPH oxidase family of reactive oxygen species (ROS) generating enzymes. Altered expression and activity of Nox5 has been reported in cardiovascular diseases and cancers but the absence of Nox5 in rodents has precluded a greater understanding of its physiological and pathophysiological roles. Multiple polymorphisms have been identified within the coding sequence of human Nox5, but whether this translates into altered enzyme function is unknown. Herein, we have generated 15 novel mutants of Nox5β to evaluate the effect of exonic SNPs on basal and stimulated enzyme activity. Compared to the WT enzyme, ROS production was unchanged or slightly modified in the majority of mutants, but significantly decreased in 7. Focusing on M77K, Nox5 activity was dramatically reduced in unstimulated cells and following challenge with both calcium- and phosphorylation-dependent stimuli despite equivalent levels of expression. The M77K mutation did not influence the Nox5 phosphorylation or the ability to bind Hsp90, but in cell-free assays with excess co-factors and calcium, ROS production was dramatically reduced. A more conservative substitution M77V arising from another SNP yielded a different profile of enzyme activity and suggests a critical role of M77 in calcium-dependent ROS production. Two C-terminal mutants, R530H and G542R, were observed that had little to no activity and relatively high minor allele frequency (MAF). In conclusion, we have identified 7 missense SNPs in Nox5 that result in little or no enzyme activity. Whether humans with dysfunctional Nox5 variants have altered physiology or disease remains to be determined

Impact of Nox5 Polymorphisms on Basal and Stimulus-Dependent ROS Generation

<div><p>Nox5 is an EF-hand containing, calcium-dependent isoform of the NADPH oxidase family of reactive oxygen species (ROS) generating enzymes. Altered expression and activity of Nox5 has been reported in cardiovascular diseases and cancers but the absence of Nox5 in rodents has precluded a greater understanding of its physiological and pathophysiological roles. Multiple polymorphisms have been identified within the coding sequence of human Nox5, but whether this translates into altered enzyme function is unknown. Herein, we have generated 15 novel mutants of Nox5β to evaluate the effect of exonic SNPs on basal and stimulated enzyme activity. Compared to the WT enzyme, ROS production was unchanged or slightly modified in the majority of mutants, but significantly decreased in 7. Focusing on M77K, Nox5 activity was dramatically reduced in unstimulated cells and following challenge with both calcium- and phosphorylation-dependent stimuli despite equivalent levels of expression. The M77K mutation did not influence the Nox5 phosphorylation or the ability to bind Hsp90, but in cell-free assays with excess co-factors and calcium, ROS production was dramatically reduced. A more conservative substitution M77V arising from another SNP yielded a different profile of enzyme activity and suggests a critical role of M77 in calcium-dependent ROS production. Two C-terminal mutants, R530H and G542R, were observed that had little to no activity and relatively high minor allele frequency (MAF). In conclusion, we have identified 7 missense SNPs in Nox5 that result in little or no enzyme activity. Whether humans with dysfunctional Nox5 variants have altered physiology or disease remains to be determined.</p></div

Sequences of Nox5 mutagenic primers.

<p>Sequences of Nox5 mutagenic primers.</p

Nonsense and missense single nucleotide polymorphisms within the gene coding region of Nox5. Position numbers refer to AF325189.1

<p>*EA: European Ameriacan.</p><p>*AA: African American.</p><p>*Reference species: <i>Homo sapiens, Bos taurus, Canis lupus familiaris, Oryctolagus cuniculus, Equus caballus, Macaca mulatta, Gorilla gorilla, Ovis aries, Felis catus and Sus scrofa.</i></p

Demographics of SNP induced mutations in Nox5.

<p>ASW: Americans of African Ancestry in SW USA.</p><p>CEU: Utah Residence (CEPH) with Western and Northern European Ancestry.</p><p>CHB: Han Chineses in Beijing, China.</p><p>CHS: Southern Han Chinese.</p><p>CLM: Colombians from Medelin, Colombia.</p><p>FIN: Finnish in Finland.</p><p>GBR: British in England and Scotland.</p><p>IBS: Iberian population in Spain.</p><p>JPT: Japanese in Tokyo, Japan.</p><p>LWK: Luhya in Webuye, Kenya.</p><p>MXL: Mexican Ancestry from Los Angeles USA.</p><p>PUR: Puerto Ricans from Puerto Rico.</p><p>TSI: Toscani in Italia.</p><p>YRI: Yoruba in Ibadan, Nigera.</p

The M77K mutation decreases the calcium-induced activation of Nox5.

<p>(<b>A</b>) Stimulated superoxide release was measured over time from COS-7 cells expressing HA-Nox5 or the mutant M77K following addition with ionomycin (1 µM). Results show the maximum level of superoxide produced and are presented as mean ± SEM (n = 4–6), *P<0.05, versus HA-Nox5. (<b>B</b>) The activity of Nox5 in cell free extracts was determined in the absence or in the presence of free calcium (mean ± SEM n = 4). *<i>P</i><0.05, <i>versus</i> HA-Nox5.</p

Basal superoxide production from Nox5 mutants.

<p>Superoxide production was monitored using L-012 in COS-7 cells transfected with HA-Nox5 or mutants based on SNPs listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100102#pone-0100102-t002" target="_blank"><b>Table 2</b></a> (A–O). Upper panel shows unstimulated or basal superoxide release from HA-Nox5 or the mutants. Western blots in the lower panels reveal full length protein expression of transgenes versus the loading control, GAPDH. Results are presented as mean ± SEM (n = 4–6), *<i>P</i><0.05, <i>versus</i> HA-Nox5.</p