Interaction between CD40, a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ligand CD40L, a 39-kDa glycoprotein, is essential for the development of humoral and cellular immune responses 1,2 . Selective blockade or activation of this pathway provides the ground for the development of new treatments against immunologically based diseases 3,4 and malignancies 5,6 . Like other members of the TNF superfamily, CD40L monomers self-assemble around a threefold symmetry axis to form noncovalent homotrimers that can each bind three receptor molecules 7,8 . Here, we report on the structure-based design of small synthetic molecules with C 3 symmetry that can mimic CD40L homotrimers. These molecules interact with CD40, compete with the binding of CD40L to CD40, and reproduce, to a certain extent, the functional properties of the much larger homotrimeric soluble CD40L. Architectures based on rigid C 3 -symmetric cores may thus represent a general approach to mimicking homotrimers of the TNF superfamily.CD40L is expressed mainly on activated T cells, whereas its cognate receptor, CD40, is constitutively expressed on dendritic cells (DC), macrophages and B cells. The engagement of CD40 by its ligand contributes to regulation of B cell proliferation, immunoglobulin production, immunoglobulin class switching, germinal center formation and development of B cell memory 1 . Moreover, CD40-CD40L interaction has an essential role in cellular immune response in which CD40 ligation activates DCs, 'licensing' them to present antigen to cytotoxic T cells by increasing MHC and costimulatory molecule expression and by producing high levels of IL-12, a T cell-stimulating cytokine [9][10][11] . Antibodies against CD40 with agonist activity have been used to increase immune response in infectious diseases 12,13 and in cancer immunotherapy 5,6 . All of these results underscore the important therapeutic applications that could emerge from the development of small-molecule CD40 agonists.Although ligand-induced dimerization is a general mechanism for activating receptors of cytokines and growth factors 14 , signaling through receptors of the TNFR superfamily strongly relies on the formation of stoichiometrically defined C 3 -symmetric complexes 7 . The structures of several TNF family members in complex with their cognate receptors show that each ligand homotrimer interacts with three monomeric receptor chains 7,15,16 . The geometry of the resulting 3:3 hexameric complex is favorable to the formation of an internal 3:3 signaling complex between the intracellular tail of the receptor and transduction proteins, ultimately activating downstream effector pathways 7 .Despite the difficulty in identifying small molecules that can disrupt protein-protein interactions, synthetic agonists of homodimeric cytokine receptors have been reported 17,18 . The ability of these molecules to dimerize cell-surface receptors is a major determinant of their effector functions 19 . In the present study, we have developed CD40L mimetics by integr...

Resveratrol, a phytoalexin found in the skin of grapes, is believed to have multiple bioactivities including anti-cancer, anti-carcinogenesis and antiinflammatory. The mechanisms by which resveratrol might produce these effects are not well understood. In this study, malignant human pancreatic cancer cells were treated without or with resveratrol in combination with ionizing radiation (IR), and then the mitochondrial function of treated cells was evaluated using several standardized assays. They include the Calcein AM method for mitochondria transition pore; the JC-1 staining method for mitochondria membrane potential; the CM-H2DCFDA method for reactive oxygen species; and the Annexin V/propidium iodide (PI) method for apoptosis/cell death. Our results indicated that (1) pore function was partially intact after resveratrol, but resveratrol probably interfered with the accumulation of intracellular Calcein AM; (2) depolarization of the mitochondria membrane was increased in the resveratrol treated cells, consistent with mitochondrial dysfunction; (3) ROS was slightly increased with resveratrol, a phenomenon that was greatly increased when this agent was combined with IR; and (4) in parallel with the above changes in mitochondrial and drug transport, cells treated with resveratrol showed increased apoptosis as measured by Annexin V/PI staining. In summary, the anti-cancer effect of resveratrol is associated with the damage of mitochondrial function that leads to increased ROS, apoptosis, and possibly intracellular drug accumulation via inhibition of proteins involved in multi-drug resistance (MDR).

Purpose: Fragile X syndrome is caused by expansion and methylation of a CGG tract in the 5Ј untranslated region of the FMR1 gene. The estimated frequency of expanded alleles (Ն55 repeats) in the United States is 1:257-1:382, but these estimates were not calculated from unbiased populations. We sought to determine the frequency of fragile X syndrome premutation (55-200 repeats) and full mutation (Ͼ200 repeats) alleles in nonselected, unbiased populations undergoing routine carrier screening for other diseases. Methods: A previously validated laboratory-developed test using triplet-primed polymerase chain reaction was used to detect premutation and full mutation alleles in an unselected series of 11,759 consecutive cystic fibrosis carrier screening samples and 2011 samples submitted for screening for genetic diseases prevalent among the Ashkenazi Jewish population. Results: Premutations were identified in 48 cystic fibrosis screening samples (1:245) and 15 samples (1:134) from the Ashkenazi Jewish population. Adjusted for the ethnic mix of the US population and self-reported ethnicity in our screening population, the estimated female premutation carrier frequency in the United States was 1:178. The calculated frequency of full mutation alleles was 1:3335 overall, and the calculated premutation frequency in males was 1:400. Based on frequency of larger, Ն70 repeat alleles, and reported penetrance, the calculated fragile X-associated tremor and ataxia syndrome, and fragile X-associated primary ovarian insufficiency frequencies is 1:4848 and 1:3560, respectively. Conclusion: Our calculated fragile X syndrome carrier rate is higher than previous estimates for the US population and warrants further consideration of population-based carrier screening. Genet Med 2011:13(1): 39 -45.

Purpose: Fragile X syndrome is caused by expansion and subsequent methylation of a CGG trinucleotide repeat in the FMR1 5Ј-untranslated region. Southern blot analysis is typically required to determine expansion size for triplet repeat lengths Ͼ200. We describe a triplet-primed polymerase chain reaction-based method using automated capillary electrophoresis detection for qualitative assessment of expanded CGG repeats. Methods: The assay uses triplet-primed polymerase chain reaction in combination with GC-melting reagents and substitution of 7-deaza-2-deoxyGTP for dGTP. Amplicons are resolved by capillary electrophoresis. Results: A distinctive pattern of tapering or "stutter" polymerase chain reaction amplification was evident on capillary electrophoresis in male and female patients harboring all expanded allele lengths examined (up to 2000 CGG repeats) and could be used to differentiate normal, intermediate, premutation, and full mutation alleles. Full mutation alleles exhibited an additional late-migrating amplicon on capillary electrophoresis. Mixing experiments demonstrated sensitivity as low as 1% for detection of the full mutation allele. In a 1275-sample concordance study against our existing polymerase chain reaction platform (with Southern blot analysis for repeat lengths Ն55), the triplet-primed polymerase chain reaction method exhibited 100% concordance for normal, intermediate, expanded, and full mutation alleles. This method also detected the full mutation alleles in DNA isolated from blood spots. Conclusion: This assay provides an accurate assessment of FMR1 repeat status and holds promise for use in carrier and newborn screening. The method distinguishes normal homozygous females from full mutation carrying females. Although the method is not useful for accurate sizing, it supplements the classic polymerase chain reaction method and results in significant reduction in the number of Southern blot analyses required to be performed in the laboratory to accurately assess the FMR1 genotype in all individuals. Genet Med 2010:12(3):162-173.

Purpose:We sought to determine the genotype frequencies for cytochrome p450 enzyme 2C19 variant alleles both in the US panethnic population and various US ethnic groups and to establish the frequency of clinically actionable genotypes. methods:Analytical results were obtained from 1,396 consecutive samples submitted for cytochrome p450 enzyme 2C19 genotyping tests and stored in a proprietary database. This database was queried and genotypes and predicted phenotypes established. Anonymized samples were obtained from specimens submitted for cystic fibrosis genotyping that contained ethnicity information. Samples from 357, 149, and 346 individuals self-identified as white, African American, and Hispanic, respectively, were analyzed. In addition, 342 anonymized samples submitted for Ashkenazi Jewish panel testing were analyzed.Results: Significant ethnic differences were observed in the frequencies of the *17 ultrarapid allele among the various groups studied. In the pan-ethnic population, 3.8% of tested patients were classified as ultrarapid metabolizers, 24% as extensive metabolizers heterozygous for a *17 ultrarapid allele, 27% as intermediate metabolizers, and 3.5% as poor metabolizers. Using stringent criteria, 7.3% of individuals would have clinically actionable genotypes. In addition, we detected two individuals with a haplotype of *2/*17 and a single individual with a haplotype of *4/*17 indicating that the *17 hypermetabolic allele can occur on a *1, *2, or *4 background.Genet Med 2012:14(1):95-100

Induction of the carcinogen-metabolizing enzyme cytochrome P4501A1 (CYP1A1) is a key step in the development of tobacco-related cancers. To determine if marijuana smoke activates CYP1A1, a murine hepatoma cell line expressing an inducible CYP1A1 gene (Hepa-1) was exposed in vitro to tar extracts prepared from either tobacco, marijuana, or placebo marijuana cigarettes. Marijuana tar induced higher levels of CYP1A1 messenger RNA (mRNA) than did tobacco tar, yet resulted in much lower CYP1A1 enzyme activity. These differences between marijuana and tobacco were primarily due to Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the psychoactive component of marijuana. Here we show that Delta(9)-THC acts through the aryl hydrocarbon receptor complex to activate transcription of CYP1A1. A 2-microg/ml concentration of Delta(9)-THC produced an average 2.5-fold induction of CYP1A1 mRNA, whereas a 10- microg/ml concentration of Delta(9)-THC produced a 4.3-fold induction. No induction was observed in Hepa-1 mutants lacking functional aryl-hydrocarbon receptor or aryl-hydrocarbon receptor nuclear translocator genes. At the same time, Delta(9)-THC competitively inhibited the CYP1A1 enzyme, reducing its ability to metabolize other substrates. Spiking tobacco tar with Delta(9)-THC resulted in a dose-dependent decrease in the ability to generate CYP1A1 enzyme activity as measured by the ethoxyresorufin-o-deethylase (EROD) assay. This inhibitory effect was confirmed by Michaelis-Menton kinetic analyses using recombinant human CYP1A1 enzyme expressed in insect microsomes. This complex regulation of CYP1A1 by marijuana smoke and the Delta(9)-THC that it contains has implications for the role of marijuana as a cancer risk factor.

Antioxidants have been studied for their capacity to reduce the cytotoxic effects of radiation in normal tissues for at least 50 years. Early research identified sulfur-containing antioxidants as those with the most beneficial therapeutic ratio, even though these compounds have substantial toxicity when given in-vivo. Other antioxidant molecules (small molecules and enzymatic) have been studied for their capacity to prevent radiation toxicity both with regard to reduction of radiation-related cytotoxicity and for reduction of indirect radiation effects including long-term oxidative damage. Finally, categories of radiation protectors that are not primarily antioxidants, including those that act through acceleration of cell proliferation (e.g. growth factors), prevention of apoptosis, other cellular signaling effects (e.g. cytokine signal modifiers), or augmentation of DNA repair, all have direct or indirect effects on cellular redox state and levels of endogenous antioxidants. In this review we discuss what is known about the radioprotective properties of antioxidants, and what those properties tell us about the DNA and other cellular targets of radiation.

The objective of this study was to design and validate a next-generation sequencing assay (NGS) to detect BRCA1 and BRCA2 mutations. We developed an assay using random shearing of genomic DNA followed by RNA bait tile hybridization and NGS sequencing on both the Illumina MiSeq and Ion Personal Gene Machine (PGM). We determined that the MiSeq Reporter software supplied with the instrument could not detect deletions greater than 9 base pairs. Therefore, we developed an alternative alignment and variant calling software, Quest Sequencing Analysis Pipeline (QSAP), that was capable of detecting large deletions and insertions. In validation studies, we used DNA from 27 stem cell lines, all with known deleterious BRCA1 or BRCA2 mutations, and DNA from 67 consented control individuals who had a total of 352 benign variants. Both the MiSeq/QSAP combination and PGM/Torrent Suite combination had 100% sensitivity for the 379 known variants in the validation series. However, the PGM/Torrent Suite combination had a lower intra- and inter-assay precision of 96.2% and 96.7%, respectively when compared to the MiSeq/QSAP combination of 100% and 99.4%, respectively. All PGM/Torrent Suite inconsistencies were false-positive variant assignments. We began commercial testing using both platforms and in the first 521 clinical samples MiSeq/QSAP had 100% sensitivity for BRCA1/2 variants, including a 64-bp deletion and a 10-bp insertion not identified by PGM/Torrent Suite, which also suffered from a high false-positive rate. Neither the MiSeq nor PGM platform with their supplied alignment and variant calling software are appropriate for a clinical laboratory BRCA sequencing test. We have developed an NGS BRCA1/2 sequencing assay, MiSeq/QSAP, with 100% analytic sensitivity and specificity in the validation set consisting of 379 variants. The MiSeq/QSAP combination has sufficient performance for use in a clinical laboratory.