A Novel Method for Fragmentation Studies in Particle Therapy: Principles of Ion Identification.

PURPOSE: In carbon ion beam radiation therapy, fragmentation processes within the patient lead to changes in the composition of the particle field with increasing depth. Consequences are alterations of the resulting dose distribution and its biological effectiveness. To enable accurate treatment planning, the characteristics of the ion spectra resulting from fragmentation processes need to be known for various ion energies and target materials. In this work, we present a novel method for ion type identification using a small and highly flexible setup based on a single detector and designed to simplify measurements and overcome current shortages in available fragmentation data. MATERIALS AND METHODS: The presented approach is based on the pixelated, semiconductor detector Timepix. The large number of pixels with small pitch, all individually calibrated for energy deposition, enables detection and visualization of single particle tracks. For discrimination among different ion species, the pattern recognition analysis of the detector signal is used. Fragmentation spectra resulting from a primary carbon ion beam at various depths of tissue-equivalent material were studied to identify different ion species in mixed particle fields. The performance of the method was evaluated quantitatively using reference data from an established technique. RESULTS: All ion species resulting from carbon ion fragmentation in tissue-equivalent material could be separated. For measurements behind a 158-mm-thick water tank, the relative fractions of H, He, Be, and B ions detected agreed with corresponding reference data within the limits of uncertainty. For the relatively rare lithium ions, the agreement was within 2.3 Δref (uncertainty of reference). CONCLUSION: For designated configurations, the presented ion type identification method enables studies of therapeutic carbon ion beams with a simple, small, and configurable detection setup. The technique is promising to enable online fragmentation studies over a wide range of beam and target parameters in the future.

Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques.

A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

Jacobsen catalyst as a cytochrome P450 biomimetic model for the metabolism of monensin A.

Monensin A is a commercially important natural product isolated from Streptomyces cinnamonensins that is primarily employed to treat coccidiosis. Monensin A selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties. In this study, we evaluated the Jacobsen catalyst as a cytochrome P450 biomimetic model to investigate the oxidation of monensin A. Mass spectrometry analysis of the products from these model systems revealed the formation of two products: 3-O-demethyl monensin A and 12-hydroxy monensin A, which are the same ones found in in vivo models. Monensin A and products obtained in biomimetic model were tested in a mitochondrial toxicity model assessment and an antimicrobial bioassay against Staphylococcus aureus, S. aureus methicillin-resistant, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli. Our results demonstrated the toxicological effects of monensin A in isolated rat liver mitochondria but not its products, showing that the metabolism of monensin A is a detoxification metabolism. In addition, the antimicrobial bioassay showed that monensin A and its products possessed activity against Gram-positive microorganisms but not for Gram-negative microorganisms. The results revealed the potential of application of this biomimetic chemical model in the synthesis of drug metabolites, providing metabolites for biological tests and other purposes.

Biosynthesis of colabomycin E, a new manumycin-family metabolite, involves an unusual chain-length factor.

Colabomycin E is a new member of the manumycin-type metabolites produced by the strain Streptomyces aureus SOK1/5-04 and identified by genetic screening from a library of streptomycete strains. The structures of colabomycin E and accompanying congeners were resolved. The entire biosynthetic gene cluster was cloned and expressed in Streptomyces lividans. Bioinformatic analysis and mutagenic studies identified components of the biosynthetic pathway that are involved in the formation of both polyketide chains. Recombinant polyketide synthases (PKSs) assembled from the components of colabomycin E and asukamycin biosynthetic routes catalyzing the biosynthesis of "lower" carbon chains were constructed and expressed in S. aureus SOK1/5-04 ΔcolC11-14 deletion mutant. Analysis of the metabolites produced by recombinant strains provided evidence that in both biosynthetic pathways the length of the lower carbon chain is controlled by an unusual chain-length factor supporting biosynthesis either of a triketide in asukamycin or of a tetraketide in colabomycin E. Biological activity assays indicated that colabomycin E significantly inhibited IL-1ß release from THP-1 cells and might thus potentially act as an anti-inflammatory agent.

In vitro metabolism of monensin A: microbial and human liver microsomes models.

1. Monensin A, an important antibiotic ionophore that is primarily employed to treat coccidiosis, selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties. 2. In this study, we evaluated the fungi Cunninghamella echinulata var. elegans ATCC 8688A, Cunninghamella elegans NRRL 1393 ATCC 10028B and human hepatic microsomes as CYP-P450 models to investigate the in vitro metabolism of monensin A and compare the products with the metabolites produced in vivo. 3. Mass spectrometry analysis of the products from these model systems revealed the formation of three metabolites: 3-O-demethyl monensin A, 12-hydroxy monensin A and 12-hydroxy-3-O-demethyl monensin A. We identified these products by tandem mass spectrometry and through comparison with the in vivo metabolites. 4. This analysis demonstrated that the model systems produce the same metabolites found in in vivo studies, thus they could be used to predict the metabolism of monensin A. Furthermore, we verified that liquid chromatography coupled to mass spectrometry is a powerful tool to study the in vitro metabolism of drugs, because it allows the successful identifications of several derivatives from different metabolic models.

Biochemical and genetic insights into asukamycin biosynthesis.

Asukamycin, a member of the manumycin family metabolites, is an antimicrobial and potential antitumor agent isolated from Streptomyces nodosus subsp. asukaensis. The entire asukamycin biosynthetic gene cluster was cloned, assembled, and expressed heterologously in Streptomyces lividans. Bioinformatic analysis and mutagenesis studies elucidated the biosynthetic pathway at the genetic and biochemical level. Four gene sets, asuA-D, govern the formation and assembly of the asukamycin building blocks: a 3-amino-4-hydroxybenzoic acid core component, a cyclohexane ring, two triene polyketide chains, and a 2-amino-3-hydroxycyclopent-2-enone moiety to form the intermediate protoasukamycin. AsuE1 and AsuE2 catalyze the conversion of protoasukamycin to 4-hydroxyprotoasukamycin, which is epoxidized at C5-C6 by AsuE3 to the final product, asukamycin. Branched acyl CoA starter units, derived from Val, Leu, and Ile, can be incorporated by the actions of the polyketide synthase III (KSIII) AsuC3/C4 as well as the cellular fatty acid synthase FabH to produce the asukamycin congeners A2-A7. In addition, the type II thioesterase AsuC15 limits the cellular level of omega-cyclohexyl fatty acids and likely maintains homeostasis of the cellular membrane.

New carbasugars from Streptomyces lincolnensis.

Two new carbasugars (9 and 10) were isolated from Streptomyces lincolnensis DSM 40355 along with streptol (valienol, 8), gabosine I (valienone, 14), and glucosylglycerate. The reported (1)H and (13)C assignments are based on 1D ((1)H, (13)C, 1D-TOCSY, homodecoupling) and 2D (gCOSY, J-resolved, TOCSY, ROESY, gHSQC, gHMBC) NMR techniques and electrospray ionization FT mass spectrometry (ESI FTMS).

HPLC-fluorescence detection method for determination of key intermediates of the lincomycin biosynthesis in fermentation broth.

The biosynthetic pathway of the clinically important antibiotic lincomycin is not known in details. The precise knowledge of the lincomycin biosynthesis is a prerequisite for generation of improved derivatives by means of combinatorial genetics. Methods allowing determination of the key intermediates are very important tools of the pathway investigation. Two new high-performance liquid chromatography methods with fluorescence detection for determination of lincomycin precursors in fermentation broth of Streptomyces lincolnensis and its lincomycin nonproducing mutants were developed. The first one enables simultaneous analysis of methylthiolincosamide (MTL) and N-demethyllincomycin (NDL), whereas the second one is suitable for 4-propyl-L-proline (PPL) assay. Both methods are based on the pre-column derivatization: MTL and NDL with 4-chloro-7-nitrobenzofurazan; PPL with o-phthaldialdehyde. The methods were validated with lower limit of quantification values of 2.50, 3.75, and 3.75 microg ml(-1) for MTL, NDL, and PPL, respectively. The inter- and intra-day accuracies and precisions were all within 12%. Stability of oxidized and derivatized analytes was investigated.

Small-scale in vivo imaging of soft tissues by radioscopy using single X-ray photon counting.

A method has been developed for routine laboratory visualisation of small-scale soft tissue by means of transmission X-ray radioscopy and tomography. Using termites as models, imaging quality with a spatial resolution of about 3 mum was achieved and 3D tomographic reconstruction was demonstrated. A termite worker individual was visualized before and after its metamorphosis towards the soldier caste. The developed methodology represents a non-invasive and real-time way of acquiring 3D anatomic data with a high contrast so that it is a promising candidate to become a tool for routine investigations in life sciences.

Extracellular carbohydrate metabolites from Streptomyces coelicolor A3(2).

A new ribose trisaccharide, alpha-Ribf-(1-->2)-alpha-Ribf-(1-->3)-alpha-Ribf (1), was isolated together with 5-O-(alpha-mannosyl)-myo-inositol (2), 2-O-(alpha-mannosyl)-myo-inositol (3), trehalose (4), and d-ribulose (5) from a submerged cultivation of Streptomyces coelicolor A3(2). The structures of these compounds were elucidated by spectroscopic and chemical methods. Concentrations of these compounds in the medium were in the range from 0.04 (1) to 0.5 (4) mg/mL.