Intralaboratory validation of cell-free translation assay for detecting ricin toxin biological activity

A cell-free translation (CFT) assay for determining ricin biological activity was validated. The statistical data from the validation study showed a high level of precision within and between runs of the assay. The assay was specific for determining ricin biological activity in food-based matrixes and discriminated ricin from other ribosome-inactivating proteins. The mean bias (relative error) between measured ricin concentrations of 3 validation samples and their nominal concentrations was 1.1, 6.6, and 20.3%, while the coefficient of variation (CV) was 14.1, 7.7, and 13.5%, respectively, demonstrating good precision, accuracy, and linearity. The CVs of ricin concentrations in 2 ricin-containing samples calculated from a dilution series were <5 and <12%, respectively, demonstrating very good parallelism. The analyte stability of ricin-containing samples stored for 1 month either at 4 or -20 degrees C, the stability of ricin stock solutions, and the results of assays executed by different analysts and using different luminometers were evaluated. The statistical validation data confirmed that the 4-parameter logistic equation, y = (a - d)/[1 + (x/c)b] + d, provided an accurate representation of a sigmoidal relationship between the measured response and the observed ricin concentration for the CFT assay.     

Precise mass measurement of a double-stranded 500 base-pair (309 kDa) polymerase chain reaction product by negative ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) has been used to determine the mass of a double-stranded 500 base-pair (bp) polymerase chain reaction (PCR) product with an average theoretical mass of the blunt-ended (i.e. unadenylated) species of 308 859.35 Da. The PCR product was generated from the linearized bacteriophage Lambda genome which is a double-stranded template. Utilization of ethanol precipitation in tandem with a rapid microdialysis step to purify and desalt the PCR product was crucial to obtain a precise mass measurement. The PCR product (0.8 pmol/μL) was electrosprayed from a solution containing 75% acetonitrile, 25 mM piperidine, and 25 mM imidazole and was infused at a rate of 200 nL/min. The average molecular mass and the corresponding precision were determined using the charge-states ranging from 172 to 235 net negative charges. The experimental mass and corresponding precision (reported as the 95% confidence interval of the mean) was 309 406 +/- 27 Da (87 ppm). The mass accuracy was compromised due to the fact that the PCR generates multiple products when using Taq polymerase due to the non-template directed 3'-adenylation. This results in a mixture of three PCR products with nearly identical mass (i.e. blunt-ended, mono-adenylated and di-adenylated) with unknown relative abundances that were not resolved in the spectrum. Thus, the experimental mass will be a weighted average of the three species which, under our experimental conditions, reflects a nearly equal concentration of the mono- and di-adenylated species. This report demonstrates that precise mass measurements of PCR products up to 309 kDa (500 bp) can be routinely obtained by ESI-FTICR requiring low femtomole amounts. Copyright 1999 John Wiley & Sons, Ltd.     

Masked imidazolyl-dipyrromethanes in the synthesis of imidazole-substituted porphyrins

Imidazole-substituted metalloporphyrins are valuable for studies of self-assembly and for applications where water solubility is required. Rational syntheses of porphyrins bearing one or two imidazol-2-yl or imidazol-4-yl groups at the meso positions have been developed. The syntheses employ dipyrromethanes, 1-acyldipyrromethanes, and 1,9-diacyldipyrromethanes bearing an imidazole group at the 5-position. The polar, reactive imidazole unit was successfully masked by use of (1) the 2-(trimethylsilyl)ethoxymethyl (SEM) group at the imidazole pyrrolic nitrogen, and (2) a dialkylboron motif bound to the pyrrole of the dipyrromethane and coordinated to the imidazole imino nitrogen. The nonpolar nature of such doubly masked imidazolyl-dipyrromethanes facilitated handling. Selected masked dipyrromethanes were characterized by 11B and 15N NMR spectroscopy. Five distinct methods were examined to obtain trans-A2B2-, trans-AB2C-, and trans-AB-porphyrins. Each porphyrin contained one or two SEM-protected imidazole units. The SEM group could be removed with TBAF or HCl. Two zinc(II) porphyrins and a palladium(II) porphyrin bearing a single imidazole moiety were prepared and subjected to alkylation (with ethyl iodide, 1,3-propane sultone, or 1,4-butane sultone) to give water-soluble imidazolium- porphyrins. This work establishes the foundation for the rational synthesis of a variety of porphyrins containing imidazole units.     

Synthetic chlorins bearing auxochromes at the 3- and 13-positions

Synthetic chlorins bearing diverse auxochromes at the 3- and 13-positions of the macrocycle are valuable targets given their resemblance to chlorophylls a and b, which bear 3-vinyl and 13-keto groups. A de novo route has been exploited to construct nine zinc chlorins bearing substituents at the 3- and 13-positions and two benchmark zinc chlorins lacking such substituents. The chlorins are sterically uncongested and bear (1) a geminal dimethyl group in the reduced pyrroline ring, (2) a H, an acetyl, a triisopropylsilylethynyl (TIPS-ethynyl), or a vinyl at the 3-position, (3) a H, an acetyl, or TIPS-ethynyl at the 13-position, and (4) a H or a mesityl at the 10-position. The synthesis of the 13-substituted chlorins relied on p-TsOH x H2O-catalyzed condensation of an 8,9-dibromo-1-formyldipyrromethane (eastern half) and 2,3,4,5-tetrahydro-1,3,3-trimethyldipyrrin (western half), followed by metal-mediated oxidative cyclization, affording the 13-bromochlorin. Similar use of a bromo- or TIPS-ethynyl-substituted western half provided access to 3-substituted chlorins. A 3-bromo, 13-bromo, or 3,13-dibromochlorin was further transformed by Pd-coupling to introduce the vinyl group (via tributylvinyltin), TIPS-ethynyl group (via TIPS-acetylene), or acetyl group (via tributyl(1-ethoxyvinyl)tin, followed by acidic hydrolysis). In the 10-mesityl-substituted zinc chlorins, the series of substituents, 3-vinyl, 13-TIPS-ethynyl, 3-TIPS-ethynyl, 13-acetyl, 3,13-bis(TIPS-ethynyl), 3-TIPS-ethynyl-13-acetyl, or 3,13-diacetyl, progressively causes (1) a redshift in the absorption maximum of the B band (405-436 nm) and the Q(y) band (606-662 nm), (2) a relative increase in the intensity of the Q(y) band (I(B)/I(Q) = 4.2-1.5), and (3) an increase in the fluorescence quantum yield phi(f) (0.059-0.29). The zinc chlorins bearing a 3-TIPS-ethynyl-13-acetyl or a 3,13-diacetyl group exhibit a number of spectral properties resembling those of chlorophyll a or its zinc analogue. Taken together, this study provides access to finely tuned chlorins for spectroscopic studies and diverse applications.     

Subtypes of medulloblastoma have distinct developmental origins

Medulloblastoma encompasses a collection of clinically and molecularly diverse tumour subtypes that together comprise the most common malignant childhood brain tumour. These tumours are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) after aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH subtype). The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT subtype) arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumours infiltrate the dorsal brainstem, whereas SHH-subtype tumours are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem which included aberrantly proliferating Zic1(+) precursor cells. These lesions persisted in all mutant adult mice; moreover, in 15% of cases in which Tp53 was concurrently deleted, they progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence, to our knowledge, that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH- and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer.     

The Preparation, Characterization and X-ray Structural Analysis of Tetrakis[1-Methyl-3-(2-Propyl)-2(3H)-Imidazolethione]Cadmium(II) Hexafluorophosphate

Abstract  A new compound, [Cd(mipit)₄][PF₆]₂ has been synthesized and characterized via standard solid and solution state methods including single crystal X-ray crystallography (mipit = 1-methyl-3-(2-propyl)-2(3H)-imidazolethione). The title compound crystallizes in tetragonal space group I4 ₁ /a with a = 12.478(2) ?, b = 12.478(2) ?, c = 28.806(6) ?, and Z = 4. The complex is a high melting, colorless solid that has a distorted tetrahedral CdS₄ coordination geometry. Thermogravimetric analysis results for the title compound as well as for another potential CdS synthon and two potential CdSe synthons are reported. Graphical Abstract  The synthesis and characterization of the title compound, a new ionic homoleptic cadmium complex, tetrakis[1-methyl-3-(2-propyl)-2(3H)-imidazolethione]cadmium(II) hexafluorophosphate is reported. The results of a single crystal X-ray diffraction analysis reveal a distorted tetrahedral CdS₄ coordination sphere about the cadmium. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biologically driven neural platform invoking parallel electrophoretic separation and urinary metabolite screening

This work reveals a computational framework for parallel electrophoretic separation of complex biological macromolecules and model urinary metabolites. More specifically, the implementation of a particle swarm optimization (PSO) algorithm on a neural network platform for multiparameter optimization of multiplexed 24-capillary electrophoresis technology with UV detection is highlighted. Two experimental systems were examined: (1) separation of purified rabbit metallothioneins and (2) separation of model toluene urinary metabolites and selected organic acids. Results proved superior to the use of neural networks employing standard back propagation when examining training error, fitting response, and predictive abilities. Simulation runs were obtained as a result of metaheuristic examination of the global search space with experimental responses in good agreement with predicted values. Full separation of selected analytes was realized after employing optimal model conditions. This framework provides guidance for the application of metaheuristic computational tools to aid in future studies involving parallel chemical separation and screening. Adaptable pseudo-code is provided to enable users of varied software packages and modeling framework to implement the PSO algorithm for their desired use.     

Nicotinamide Adenine Dinucleotide Oxidation Studies at Multiwalled Carbon Nanotube/Polymer Composite Modified Glassy Carbon Electrodes

NADH oxidation has previously been investigated at carbon nanotube surfaces, although studies into the effect of the polymer binders are needed to fully understand whether the polymer binder affects the electrochemistry. This work details NADH oxidation at glassy carbon electrodes modified with composites containing multiwalled carbon nanotubes and selected polymer binders. NADH is shown to be oxidized at a lower potential than at glassy carbon electrodes and the oxidation potential is a function of the polymer binder. Hydrophobically modified Nafion, Nafion, linear poly(ethylenimine) (LPEI), octyl‐modified LPEI, and poly(vinylpyridine) binders were studied. Experiments showed the peak current and electrochemically assessible electrode area are dependent on the polymer binder. Overall, this paper shows that polymer binders affect NADH oxidation potential at carbon nanotube modified electrodes.     

Unusual cycloadditions of o-quinone methides with oxazoles

Unusual reactions between various electron-rich oxazoles and ortho-quinone methides is described. This combination leads to some interesting adducts.     

Reference materials for PAHs in foodstuffs: results of a preliminary intercomparison of methods in experienced laboratories

Summary A preliminary intercomparison of methods which can be applied to the determination of trace amounts of PAHs in coconut oil and green kale samples has been made under an interlaboratory cooperative analytical programme organised by the Community Bureau of Reference. The techniques which were utilised were glass-capillary gas chromatography with flame-ionisation or mass spectrometric detection, and high-performance liquid chromatography with fluorescence and/or ultra-violet detection. Analyses were made for five environmentally important PAHs and the results, the methods and some associated problems are discussed.

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Referenzmaterialien für PAKs in Lebensmitteln: Ergebnisse eines vorlÄufigen Ringversuchs erfahrener Laboratorien