Fluorescence of Naphthol AS-MX is Readily Detectable in Dioxane Mixtures

Numerous enzymes have been demonstrated to be active in non-aqueous solutions, yet the utility of phosphatases under such conditions has been difficult to determine. Here, we demonstrate the ability to fluorescently detect naphthol AS‑MX in high percentages 1,4-dioxane with a fluorescence differential compared with naphthol AS‑MX phosphate. While intensities and maximum fluorescence wavelengths changed depending on solvent conditions, these results demonstrate this system’s potential for testing phosphatase activity in high amounts of dioxane

In‐situ extraction and impregnation of black walnut husk into polyethylene film using supercritical carbon dioxide with an ethanol modifier

Walnuts are commonly cultivated for their kernel, which is a rich source of antioxidant phenolic compounds. The husk likewise contains antioxidant and antimicrobial compounds, but is typically discarded without further processing. Antioxidant compounds are useful in creating active packaging films, but typically decompose at melt extrusion temperatures in polymer processing. Due to carbon dioxide\u27s low critical point and ability to swell polymer films, supercritical carbon dioxide may be used to impregnate phenolic compounds into polymers. For this study, a novel technique is used to simultaneously produce walnut husk extracts and impregnate the extract into polymer films in the same batch extractor using supercritical carbon dioxide with a 15 wt-% ethanol modifier at 60°C at 19.4 MPa. The effect of varying the loading of walnut husk in the extractor upon impregnation mass was evaluated with the impregnation mass of the film increasing with walnut husk loading. It was determined by FTIR, as well as the eduction of the protein cytochrome c, that antioxidant compounds may be extracted from walnut husks and impregnated into low-density polyethylene film (LDPE) by this technique

Identification of novel epithelial ovarian cancer loci in women of African ancestry.

Women of African ancestry have lower incidence of epithelial ovarian cancer (EOC) yet worse survival compared to women of European ancestry. We conducted a genome-wide association study in African ancestry women with 755 EOC cases, including 537 high-grade serous ovarian carcinomas (HGSOC) and 1,235 controls. We identified four novel loci with suggestive evidence of association with EOC (p < 1 × 10-6 ), including rs4525119 (intronic to AKR1C3), rs7643459 (intronic to LOC101927394), rs4286604 (12 kb 3' of UGT2A2) and rs142091544 (5 kb 5' of WWC1). For HGSOC, we identified six loci with suggestive evidence of association including rs37792 (132 kb 5' of follistatin [FST]), rs57403204 (81 kb 3' of MAGEC1), rs79079890 (LOC105376360 intronic), rs66459581 (5 kb 5' of PRPSAP1), rs116046250 (GABRG3 intronic) and rs192876988 (32 kb 3' of GK2). Among the identified variants, two are near genes known to regulate hormones and diseases of the ovary (AKR1C3 and FST), and two are linked to cancer (AKR1C3 and MAGEC1). In follow-up studies of the 10 identified variants, the GK2 region SNP, rs192876988, showed an inverse association with EOC in European ancestry women (p = 0.002), increased risk of ER positive breast cancer in African ancestry women (p = 0.027) and decreased expression of GK2 in HGSOC tissue from African ancestry women (p = 0.004). A European ancestry-derived polygenic risk score showed positive associations with EOC and HGSOC in women of African ancestry suggesting shared genetic architecture. Our investigation presents evidence of variants for EOC shared among European and African ancestry women and identifies novel EOC risk loci in women of African ancestry

Genetic Data from Nearly 63,000 Women of European Descent Predicts DNA Methylation Biomarkers and Epithelial Ovarian Cancer Risk

DNA methylation is instrumental for gene regulation. Global changes in the epigenetic landscape have been recognized as a hallmark of cancer. However, the role of DNA methylation in epithelial ovarian cancer (EOC) remains unclear. In this study, high-density genetic and DNA methylation data in white blood cells from the Framingham Heart Study (N = 1,595) were used to build genetic models to predict DNA methylation levels. These prediction models were then applied to the summary statistics of a genome-wide association study (GWAS) of ovarian cancer including 22,406 EOC cases and 40,941 controls to investigate genetically predicted DNA methylation levels in association with EOC risk. Among 62,938 CpG sites investigated, genetically predicted methylation levels at 89 CpG were significantly associated with EOC risk at a Bonferroni-corrected threshold of P <7.94 x 10(-7). Of them, 87 were located at GWAS-identified EOC susceptibility regions and two resided in a genomic region not previously reported to be associated with EOC risk. Integrative analyses of genetic, methylation, and gene expression data identified consistent directions of associations across 12 CpG, five genes, and EOC risk, suggesting that methylation at these 12 CpG may influence EOC risk by regulating expression of these five genes, namely MAPT, HOXB3, ABHD8, ARHGAP27, and SKAP1. We identified novel DNA methylation markers associated with EOC risk and propose that methylation at multiple CpG may affect EOC risk via regulation of gene expression. Significance: Identification of novel DNA methylation markers associated with EOC risk suggests that methylation at multiple CpG may affect EOC risk through regulation of gene expression.Peer reviewe

Evaluation of vitamin D biosynthesis and pathway target genes reveals UGT2A1/2 and EGFR polymorphisms associated with epithelial ovarian cancer in African American Women.

An association between genetic variants in the vitamin D receptor (VDR) gene and epithelial ovarian cancer (EOC) was previously reported in women of African ancestry (AA). We sought to examine associations between genetic variants in VDR and additional genes from vitamin D biosynthesis and pathway targets (EGFR, UGT1A, UGT2A1/2, UGT2B, CYP3A4/5, CYP2R1, CYP27B1, CYP24A1, CYP11A1, and GC). Genotyping was performed using the custom-designed 533,631 SNP Illumina OncoArray with imputation to the 1,000 Genomes Phase 3 v5 reference set in 755 EOC cases, including 537 high-grade serous (HGSOC), and 1,235 controls. All subjects are of African ancestry (AA). Logistic regression was performed to estimate odds ratios (OR) and 95% confidence intervals (CI). We further evaluated statistical significance of selected SNPs using the Bayesian False Discovery Probability (BFDP). A significant association with EOC was identified in the UGT2A1/2 region for the SNP rs10017134 (per allele OR = 1.4, 95% CI = 1.2-1.7, P = 1.2 × 10-6 , BFDP = 0.02); and an association with HGSOC was identified in the EGFR region for the SNP rs114972508 (per allele OR = 2.3, 95% CI = 1.6-3.4, P = 1.6 × 10-5 , BFDP = 0.29) and in the UGT2A1/2 region again for rs1017134 (per allele OR = 1.4, 95% CI = 1.2-1.7, P = 2.3 × 10-5 , BFDP = 0.23). Genetic variants in the EGFR and UGT2A1/2 may increase susceptibility of EOC in AA women. Future studies to validate these findings are warranted. Alterations in EGFR and UGT2A1/2 could perturb enzyme efficacy, proliferation in ovaries, impact and mark susceptibility to EOC.Includes NIHR and CRUK

Ligand-induced propagation of entropic signals across proteins

Protein-ligand interactions are of paramount importance to the field of biochemistry. They are ubiquitous across biological systems and are at the core of structural, enzymatic and signaling processes. While much is known, only recently have the nature and role of the entropic component of these protein-ligand binding events been of concern. As entropy can be defined by the number of possible microstates, measures of dynamics can be used to proxy for protein conformational entropy. This dissertation utilizes nuclear magnetic resonance (NMR)-based nuclear spin relaxation experiments to illuminate site-resolved changes in backbone (viewed through N-H amide relaxation) and side-chain (viewed through CH2D deuterium or CHD2 carbon relaxation) motions for two protein-ligand systems: the model enzyme hen egg white lysozyme binding to a small, inhibitory sugar (chitotriose) and the GTPase Cdc42Hs binding to a 46-residue peptide derived from a p21-activating kinase (PBD46). Notably, both systems demonstrate a dependence of the variance of motional change upon ligand binding on the distance to the bound ligand. Moreover, a general distance dependence is seen across a variety of protein-ligand interactions described in the literature. Upon binding a ligand, the precise directionality and magnitude in the dynamical change of each methyl probe is context-dependent and appears to have complex underpinnings, but the variance of the dynamical response falls off with the distance from the binding interface. This decay fits to a double exponential function, indicating that both a fast-decaying local response and a slow-decaying long-range response appear to be present. The existence of this slower decay rate suggests that thermodynamic motional coupling can propagate across entire proteins and potentially be utilized by nature for allosteric-type processes. Individual proteins appear to take advantage of this property in different ways, seemingly to optimize biological function, as illustrated by the bifurcation of dynamical changes in Cdc42Hs upon binding PBD46 and the core rigidification in HEWL in response to binding chitotriose. This work provides evidence of the raw material necessary for dynamically-mediated protein allostery, and therefore illustrates the potential relevance of and emphasizes the need for consideration of fast timescale methyl motions when studying protein-ligand thermodynamics

Identification of individual components of a commercial wheat germ acid phosphatase preparation.

Wheat germ acid phosphatase (WGAP) is a commercial preparation of partially purified protein commonly used in laboratory settings for non-specific enzymatic dephosphorylation. It is known that these preparations contain multiple phosphatase isozymes and are still relatively crude. This study therefore aimed to identify the protein components of a commercial preparation of wheat germ acid phosphatase using mass spectroscopy and comparative genomics. After one post-purchase purification step, the most prevalent fifteen proteins in the mixture included heat shock proteins, beta-amylases, glucoseribitol dehydrogenases, enolases, and an aminopeptidase. While not among the most abundant components, eight unique dephosphorylation enzymes were also present including three purple acid phosphatases. Furthermore, it is shown that some of these correspond to previously isolated isozymes; one of which has been also previously shown by transcriptome data to be overexpressed in wheat seeds. In summary, this study identified the major components of WGAP including phosphatases and hypothesizes the most active components towards a better understanding of this commonly used laboratory tool

5,7,3′,4′-Hydroxy substituted flavonoids reduce the heme of cytochrome c with a range of rate constants

Flavonoids are antioxidants known to be abundant in edible plants. Seven 5,7,3′,4′-tetrahydroxy substituted flavonoids representing each major flavonoid class were used as cytochrome c reductants to systematically investigate the redox role of their C-rings. Additional examples of flavonoids and benzenediols were investigated to better understand the role of the B-ring. Pseudo-first order rate constants (k obs )and apparent bimolecular rate constants (k app )values were calculated from spectroscopic measurements. Of the seven flavonoids tested, five yielded measurable observed reduction rate constants. Butein (a chalcone)had the highest apparent bimolecular rate constant (k app ), followed by taxifolin (a flavanonol), catechin (a flavanol), eriodictyol (a flavanone), and luteolin (a flavone). Quercetin (a flavonol)and cyanidin (an anthocyanidin), however, reduced cytochrome c but k app rate constants were unable to be calculated. Neither this trend nor trends in observed rate constants correlated with flavonoid pK a , solvent accessible surface area, polar surface area, reduction potential, antioxidant ability, resonance, or radical scavenging efficiency. Weak correlation, however, was found with degrees of freedom and the number of redox involved electrons. While some cytochrome c reduction rates have been reported, this study is the first to systematically investigate the role of the structure of the flavonoid C-ring across a full set of flavonoids with identical B-rings