Synthesis and biological evaluation of 3'-carboranyl thymidine analogues

Boron neutron capture therapy (BNCT) is a chemoradio-therapeutic method for the treatment of cancer. It depends on the selective targeting of tumor cells by boron-containing compounds. One category of BNCT agents with potential to selectively target tumor cells may be thymidine derivatives substituted at the 3'-position with appropriate boron moieties. Thus, several thymidine analogues were synthesized with a carborane cluster bound to the 3'-position either through an ether or a carbon linkage. The latter are the first reported carborane-containing nucleosides in which the carboranyl entity is directly linked to the carbohydrate portion of the nucleoside by a carbon-carbon bond. Low but significant phosphorylation rates in the range of 0.18% that of thymidine were observed for the carbon-linked 3'-carboranyl thymidine analogues in phosphoryl transfer assays using recombinant preparations of thymidine kinases 1 (TK1) and thymidine kinases 2 (TK2). Some of the ether-linked 3'-carboranyl thymidine analogues appeared to be slightly unstable under acidic as well as phosphoryl transfer assay conditions and were, if at all, poor substrates for TK1.     

Association of a CXCL9 polymorphism with pediatric Crohn's disease

Genetic and environmental factors contribute to the etiopathogenesis of Crohn's disease (CD) and ulcerative colitis (UC). To identify new susceptibility genes, we determined the mRNA expression level of 88 genes from different biological contexts on colonic biopsies of CD and UC patients. We show that CXCL9 was overexpressed in colonic tissue of 3/5 CD and 3/3 UC patients compared to healthy controls. SNP genotyping for the 77147452G-->A polymorphism of the CXCL9 gene on 114 pediatric IBD patients and 120 ethnically matched unaffected adults detected a minor allele frequency of 20.3% in CD patients compared to 31.3% in controls (p=0.016). Strikingly, children with homozygosity for the wild-type allele had a significant earlier onset of CD than heterozygous individuals (11.1 versus 13.8 years). This is the first report of inverse association of the CXCL9 77147452G-->A polymorphism with pediatric CD. Our data may contribute to a better understanding of the pathophysiology underlying CD.     

Can we measure ecological sustainability? Landscape pattern as an indicator for naturalness and land use intensity at regional,national and European level

European landscapes have been shaped over the centuries by processes related to human land use, which are reflected in regionally distinct landscape patterns. Since landscape pattern has been linked to biodiversity and other ecological values of the landscapes, this paper explores landscape pattern as a tool for ecological sustainability assessments at the regional (Austrian Cultural Landscapes), national (Austria) and European (European Union + Norway, Switzerland) level with focus on agricultural landscapes. A set of landscape metrics served as a basis to assess naturalness and geometrisation of Austrian and European landscapes as a proxy for their sustainability. To achieve an accurate spatially explicit assessment, we applied a spatial reference framework consisting in units that are homogeneous in biophysical and socio-economic contexts, adapted the regional approach for its application at European level, and developed relative sustainability thresholds for the landscape metrics. The analyses revealed that several landscape metrics, particularly the “Number of Shape Characterising Points” showed a high correlation with the degree of naturalness. The sustainability map of Austria based on an ordinal regression model revealed well-known problem regions of ecological sustainability. At the European level, the relative deviation from the average pattern showed clearly the simplification processes in the landscapes. However, a better spatial resolution of land cover data would add to the refinement of pattern analysis in regions and therefore the assessment of sustainability. We recommend the combination of information of different scales for the formulation and implementation of sustainability policies.     

Protein disulfide isomerase isomerizes non-native disulfide bonds in human proinsulin independent of its peptide-binding activity

Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba'c) where the b' domain was partially deleted, thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba'c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba'c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.     

Infection pressure of human alveolar echinococcosis due to village and small town foxes (<Emphasis Type="Italic">Vuples vulpes</Emphasis>) living in close proximity to residents

This study investigated the epidemiological and ecological factors to assess the infection pressure of alveolar echinococcosis to human which are living in villages and small towns. Foxes and fox faeces were examined for Echinococcus multilocularis and foxes were observed by radio telemetry in Upper Bavaria, Germany. Forty-three percent of the village foxes (n = 65) had been infected with E. multilocularis. This prevalence rate did not differ significantly from the prevalence among rural foxes, which was 39% (n = 33; χ ² = 0.12, df = 1, p = 0.727) determined by the intestinal scraping technique. PCR analyses of fox faeces showed a higher infection rate of 35% (n = 26) among rural foxes than among foxes in villages and small towns (26%, n = 69; χ ² = 0.68, df = 1, p = 0.411). One quarter of the fox faecal samples come from private gardens of residents. The radio-tracking study on 17 foxes showed that foxes preferred the built-up area and grassland outside the villages. Village foxes concentrated their activity within a range of 500 m around the settlement. Sixty-four percent of all bearings for radio-tracked foxes showed positions in areas outside the town, and 36% of bearings were within the settlement. Village foxes, which are infected with E. multilocularis, are able to carry the parasite continuously into settlements and fox faeces present an immediate source of infection to humans, especially within their gardens. Therefore, foxes are responsible for environmental E. multilocularis egg contamination in the vicinity of humans, leading to an infection risk to inhabitants of villages and small towns.     

Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production.     

A new fast method for nanoLC-MALDI-TOF/TOF-MS analysis using monolithic columns for peptide preconcentration and separation in proteomic studies

A new fast method for identification and characterization of proteolytic digests of proteins by monolithic liquid chromatography coupled with mass spectrometry has been developed. The advantages of the monolithic columns are a high-pressure stability and low back pressure resulting in higher flow rates for capillary or nanosize columns simplifying the system handling. As was shown in several publications, such monolithic stationary phases are highly qualified for the analysis of peptides and proteins, but so far, only small volumes could be injected into the system, which might hamper the sample preparation leading to protein precipitation and partial loss of sample. To overcome the problem of small injection volumes, we established a system including a short monolithic trap column to allow preconcentration of the peptides. The injected sample is flushed at higher flow rates onto the trap column, bound to the stationary phase, and in this way concentrated in a few nanoliters before starting the separation. The expanded system was optimized and tested using different reference protein samples. Eluting peptides were detected by MALDI-TOF/TOF-MS and identified by database searching. The system is now a permanent part for proteome analysis in our lab, and as such, it was successfully applied for the detection of post-translational modifications and the analysis of membrane proteins. One example for these analyses is also included in this paper.     

Cultivation of Autotrophic Ammonia-Oxidizing Archaea from Marine Sediments in Coculture with Sulfur-Oxidizing Bacteria

The role of ammonia-oxidizing archaea (AOA) in nitrogen cycling in marine sediments remains poorly characterized. In this study, we enriched and characterized AOA from marine sediments. Group I.1a crenarchaea closely related to those identified in marine sediments and “Candidatus Nitrosopumilus maritimus” (99.1 and 94.9% 16S rRNA and amoA gene sequence identities to the latter, respectively) were substantially enriched by coculture with sulfur-oxidizing bacteria (SOB). The selective enrichment of AOA over ammonia-oxidizing bacteria (AOB) is likely due to the reduced oxygen levels caused by the rapid initial growth of SOB. After biweekly transfers for ca. 20 months, archaeal cells became the dominant prokaryotes (>80%), based on quantitative PCR and fluorescence in situ hybridization analysis. The increase of archaeal 16S rRNA gene copy numbers was coincident with the amount of ammonia oxidized, and expression of the archaeal amoA gene was observed during ammonia oxidation. Bacterial amoA genes were not detected in the enrichment culture. The affinities of these AOA to oxygen and ammonia were substantially higher than those of AOB. [¹³C]bicarbonate incorporation and the presence and activation of genes of the 3-hydroxypropionate/4-hydroxybutyrate cycle indicated autotrophy during ammonia oxidation. In the enrichment culture, ammonium was oxidized to nitrite by the AOA and subsequently to nitrate by Nitrospina-like bacteria. Our experiments suggest that AOA may be important nitrifiers in low-oxygen environments, such as oxygen-minimum zones and marine sediments.Archaea have long been known as extremophiles, since most cultivated archaeal strains were cultivated from extreme environments, such as acidic, hot, and high-salt environments. The view of archaea as extremophiles (i.e., acidophiles, thermophiles, and halophiles) has radically changed by the application of molecular technologies, including PCR in environmental microbiology. Using Archaea-specific PCR primers, novel archaeal 16S rRNA gene sequences were discovered in seawater (23, 27). Following these discoveries, an ever-increasing and unexpectedly high variety of archaeal 16S rRNA gene sequences has been reported from diverse “nonextreme” environments (67). This indicates that archaea are, like bacteria, ubiquitous in the biosphere rather than exclusively inhabiting specific extreme niches. Archaea are abundant in water columns of some oceanic provinces (33, 36) and deep-subsea floor sediments (11, 12, 48). Despite the increasing number of reports of the diversity and abundance of these nonextreme archaea by molecular ecological studies, their physiology and ecological roles have remained enigmatic.Oxidation of ammonia, a trait long thought to be exclusive to the domain Bacteria (13), was recently suggested to be a trait of archaea of the crenarchaeal groups I.1a and I.1b, based on a metagenome analysis (79) and supported by the discovery of archaeal amoA-like genes in environmental shotgun sequencing studies of Sargasso Sea water (80) and genomic analysis of “Candidatus Cenarchaeum symbiosum,” a symbiont of a marine sponge (30). Molecular ecological studies indicated that these ammonia-oxidizing archaea (AOA) are often predominant over ammonia-oxidizing bacteria (AOB) in ocean waters (9, 53, 87), soils (17, 47), and marine sediments (61). Critical evidence for autotrophic archaeal ammonia oxidation was obtained by the characterization of the first cultivated mesophilic crenarchaeon (group I.1a), “Candidatus Nitrosopumilus maritimus SCM1,” from an aquarium (38), and a related archaeon from North Sea water (87) and subsequently by enrichment of thermophilic AOA (22, 31). Whole-genome-based phylogenetic studies recently indicated that the nonthermophilic crenarchaea, including the AOA, likely form a phylum separate from the Crenarchaeota and Euryarchaeota phyla (15, 16, 72). This proposed new phylum was called Thaumarchaeota (15).Microorganisms in marine sediments contribute significantly to global biogeochemical cycles because of their abundance (85). Nitrification is essential to the nitrogen cycle in marine sediments and may be metabolically coupled with denitrification and anaerobic ammonium oxidation, resulting in the removal of nitrogen as molecular nitrogen and the generation of greenhouse gases, such as nitrous oxide (19, 75). Compared with studies on archaeal nitrification in the marine water column, only limited information on archaeal nitrification in marine sediments is available so far. Archaeal amoA genes have been retrieved from marine and coastal sediments (8, 26, 61), and the potentially important role of AOA in nitrification has been suggested based on the abundance of archaeal amoA genes relative to that of bacterial amoA genes in surface marine sediments from Donghae (South Korea) (61). Cultivation of AOA, although difficult (38), remains essential to estimating the metabolic potential of archaea in environments such as soils (47) and marine sediments (61). Here, we report the successful enrichment of AOA of crenarchaeal group I.1a from marine sediments by employing a coculture with sulfur-oxidizing bacteria (SOB) which was maintained for ca. 20 months with biweekly transfers. In this way, we were able to characterize AOA from marine sediments, providing a clue for the role of AOA in the nitrogen cycle of marine sediments.