Development and Characterization of a Monoclonal Antibody against Ochratoxin B and Its Application in ELISA

A monoclonal antibody specific to ochratoxin B (OTB) was employed for the development of an indirect competitive OTB-ELISA. The optimized OTB-ELISA resulted in a limit of detection (LOD) for OTB of 3 µg/L (8 nM), a limit of quantification (LOQ) of 3.7 µg/L (10 nM), and a 50% inhibitory concentration (IC50) of 150 nM. Due to very low cross-reactivity to OTA (2.7%) and structurally related molecules (0%), this OTB-ELISA was found to be suitable to detect OTB with excellent precision in different matrices, i.e., beer, coffee and wine. Therefore, this OTB-ELISA will allow screening of OTB in food and feed products

Structural and biochemical impact of C8-aryl-guanine adducts within the Narl recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity

Sherpa Romeo green journal, open accessChemical mutagens with an aromatic ring system may be enzymatically transformed to afford aryl radical species that preferentially react at the C8-site of 2 -deoxyguanosine (dG). The resulting carbonlinked C8-aryl-dG adduct possesses altered biophysical and genetic coding properties compared to the precursor nucleoside. Described herein are structural and in vitro mutagenicity studies of a series of fluorescent C8-aryl-dG analogues that differ in aryl ring size and are representative of authentic DNA adducts. These structural mimics have been inserted into a hotspot sequence for frameshift mutations, namely, the reiterated G3-position of the NarI sequence within 12mer (NarI(12)) and 22mer (NarI(22)) oligonucleotides. In the NarI(12) duplexes, the C8- aryl-dG adducts display a preference for adopting an anti-conformation opposite C, despite the strong syn preference of the free nucleoside. Using the NarI(22) sequence as a template for DNA synthesis in vitro, mutagenicity of the C8-aryl-dG adducts was assayed with representative high-fidelity replicative versus lesion bypass Y-family DNA polymerases, namely, Escherichia coli pol I Klenow fragment exo− (Kf−) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). Our experiments provide a basis for a model involving a two-base slippage and subsequent realignment process to relate the miscoding properties of C-linked C8-aryl-dG adducts with their chemical structures.Ye

Rectal Administration of Carbamazepine Suspension

INTRODUCTIONClinicians sometimes face a situation in which a patient requires anticonvulsant medication but is unable to take medication orally. The parenteral route may be a suitable alternative for the delivery of many medications, but may not be appropriate in other cases, for example, because of lack of IV access or patient discomfort. Furthermore, not all anticonvulsant medications are available in parenteral form. The rectal route has been used for interim or long-term administration of anticonvulsants such as diazepam,1 valproate,2,3 phenobarbital,4 carbamazepine,5-9 and (with less success) phenytoin.10-12 The clinical usefulness and bioavailability of rectally administered carbamazepine (in suspension, suppository, or gel form) has been previously described.5-9 However, very little, if any, information has been published regarding the rectal use of the commercially available Tegretol suspension, which has been available on the Canadian market since 1995. This case report describes the successful use of Tegretol suspension, administered rectally in a patient with an uncontrolled seizure disorder

Visible Fluorescent Light-up Probe for DNA Three-Way Junctions Provides Host−Guest Biosensing Applications

DNA three-way junctions (3WJs) consist of a Y-shaped hydrophobic branch point connecting three double-stranded stems and are viewed as druggable targets for cancer treatment. They are also important building blocks for the construction of DNA nanostructures and serve as recognition elements for DNA aptasensors for a wide variety of diagnostic applications. However, visible fluorescent light-up probes for specific staining of DNA 3WJs are currently lacking. Herein, we report that a merocyanine containing the N-methylbenzothiazolium (Btz) acceptor vinyl linked to a 2-fluorophenolic (FPhO) donor (FPhOBtz) serves as a universal fluorescent turn-on dye for DNA 3WJs. Our evidence is based on a multifaceted approach to define the specificity and affinity of FPhOBtz for 3WJ DNA aptamers; the cocaine binding aptamer MN4, the cholic acid binding aptamer (CABA), and four steroid aptamers (DOGS.1, DISS.1, BES.1, DCAS.1). FPhOBtz exhibits impressive turn-on (up to 730-fold) fluorescence at 580 nm upon aptamer binding with low micromolar affinity. Direct FPhOBtz displacement from the 3WJ binding domain through competitive alkaloid and steroid binding provides immediate fluorescent read out for host−guest detection strategies in human blood serum in the low micromolar regime. Our results present the first visible light-up fluorescent probe for DNA 3WJ detection strategies

Chlorine Functionalization of a Model Phenolic C8-Guanine Adduct Increases Conformational Rigidity and Blocks Extension by a Y‑Family DNA Polymerase

Certain phenoxyl radicals can attach covalently to the C8-site of 2′-deoxyguanosine (dG) to afford oxygen-linked C8-dG adducts. Such O-linked adducts can be chemically synthesized through a nucleophilic displacement reaction between a phenolate and a suitably protected 8-Br-dG derivative. This permits the generation of model O-linked C8-dG adducts on scales suitable for insertion into oligonucleotide substrates using solid-phase DNA synthesis. Variation of the C8-aryl moiety provides an opportunity to derive structure–activity relationships on adduct conformation in duplex DNA and replication bypass by DNA polymerases. In the current study, the influence of chlorine C8-dG functionalization on <i>in vitro</i> DNA replication by Klenow fragment exo^– (Kf^–) and the Y-family polymerase (Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)) has been determined. Model O-linked C8-dG adducts derived from the pentachlorophenoxyl radical ([PCP]­G) and 2,4,6-trichlorophenoxyl radical ([TCP]­G) were inserted into the reiterated G3-position of the <i>Nar</i>I sequence (12-mer, <i>Nar</i>I­(12); and 22-mer, <i>Nar</i>I­(22)), which is a known hotspot for frameshift mutations mediated by N-linked polycyclic C8-dG adducts in bacterial mutagenesis. Within the <i>Nar</i>I­(12) duplex, the unsubstituted C8-phenoxy-dG ([PhO]­G) adduct adopts a minimally perturbed B-form helix. Chlorination of [PhO]­G to afford [PCP]­G does not significantly change the adduct conformation within the <i>Nar</i>I­(12) duplex, as predicted by molecular dynamics simulations. However, when using <i>Nar</i>I­(22) for DNA synthesis <i>in vitro</i>, the chlorinated [PCP]­G and [TCP]­G lesions significantly block DNA replication by Kf^– and Dpo4, whereas [PhO]­G is readily bypassed. These findings highlight the impact that chlorine substituents impart to bulky C8-dG lesions