Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation

Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOM IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins.     

Total synthesis of citrafungin A

The antifungal natural product citrafungin A was synthesized using, as key steps, an asymmetric aldol reaction of a chiral oxazolidinone, diastereoselective alkylation of a chiral 1,3-dioxolan-2-one, semihydrogenation of an enyne, and selective methyl ester deprotection.     

Quinolinone and pyridopyrimidinone inhibitors of DNA-dependent protein kinase

8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones with selected aryl and heteroaryl groups as the substituent have been synthesised as potential inhibitors of DNA-dependent protein kinase. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was utilised in the preparation of 8-substituted 2-morpholin-4-yl-quinolin-4-ones. For this purpose 8-bromo-2-morpholin-4-yl-quinolin-4-one was required as an intermediate. This compound was obtained by adapting a literature route in which thermal cyclocondensation of (2-bromoanilino)-morpholin-4-yl-5-methylene-2,2-dimethyl[1,3]dioxane-4,6-dione afforded 8-bromo-2-morpholin-4-yl-quinolin-4-one. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was also utilised to prepare 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones using 9-hydroxy-2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-one O-trifluoromethanesulfonate as an intermediate. 8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones were both inhibitors of DNA-dependent protein kinase. When the substituent was dibenzothiophen-4-yl, dibenzofuran-4-yl or biphen-3-yl, IC50 values in the low nanomolar range were observed. Interestingly, the pyridopyrimidinones and quinolinones were essentially equipotent with the corresponding 8-substituted 2-morpholin-4-yl-chromen-4-ones previously reported (I. R. Hardcastle, X. Cockcroft, N. J. Curtin, M. Desage El-Murr, J. J. J. Leahy, M. Stockley, B. T. Golding, L. Rigoreau, C. Richardson, G. C. M. Smith and R. J. Griffin, J. Med. Chem., 2005, 48, 7829-7846).     

Determination,by inductively coupled plasma mass spectrometry,of changes in cellular metal content resulting from herpes simplex virus-1 (HSV-1) infection

Metals and metal-containing compounds are known to play important roles in many biological processes, including metabolic and detoxification pathways and the formation and function of proteins. Like all organisms, viruses are expected to contain different metals. These metals, either by themselves or in the form of metalloproteins, may be involved in the virus’s ability to infect healthy cells and replicate within them. Identification and speciation of metals in control cells and in cells affected by a virus could be helpful in elucidating infection and replication mechanisms; these might, in turn, be vital to the development of more effective treatments. There has, however, been no extensive investigation of the metals specific viruses contain or affect. The objective of this study was to investigate changes in cellular metal content resulting from herpes simplex virus 1 (HSV-1) infection. Inductively coupled plasma mass spectrometry was used to identify differences between metal concentrations in uninfected and HSV-1-infected mammalian cells. Although it can be assumed that decreases in metal content are a result of cellular response to the virus, increases can be attributed either to cellular response or to the HSV-1 virus itself. Microwave digestion and flow injection methods suitable for small sample volumes were used, and the effects of different virus inactivation procedures were explored. This work is the first step in the identification of metals pertinent to HSV-1 infection and lays the foundation for future studies concentrating on characterization of these metal-associated or containing molecules.     

Random inscribing polytopes

For convex bodies K with boundary in , we explore random polytopes with vertices chosen along the boundary of K. In particular, we determine asymptotic properties of the volume of these random polytopes. We provide results concerning the variance and higher moments of this functional, as well as an analogous central limit theorem.     

Synthesis,structure, and phase behavior of carbosilane LC dendrimers with terminal butoxyphenylbenzoate mesogenic groups

Two series of carbosilane LC dendrimers with terminal protonated and deuterated butoxyphenylbenzoate mesogenic groups linked to carbosilane dendritic matrices of the first to fifth generations via an undecylene spacer have been synthesized. The chemical structure of new dendrimers has been studied by ¹H NMR spectroscopy and gel-permeation chromatography. The dendrimers of first-fourth generations are characterized by formation of the smectic C mesophase in a wide temperature range, whereas much more complex columnar supramolecular structures are formed in dendrimers of the fifth generation. Structural studied of mesophases by X-ray diffraction and small-angle neutron scattering show that segregation takes place in mixtures of deuterated and protonated LC dendrimers; as a result, huge aggregates composed of hundreds of chemically unbound molecules develop and the sizes of these aggregates reversibly change with temperature.     

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.