An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside.

Acyclic nucleoside analogues with carboxamido- or nitro-substituted heterocyclic bases have been evaluated for their possible use as universal bases in oligodeoxynucleotides. The acyclic moiety endows the constructs with enough flexibility to allow good base stacking. The 5-nitroindazole analogue afforded the most stable duplexes among the acyclic derivatives with the least spread in Tm versus the four natural bases. In spite of the acyclic moiety, stabilities are comparable with those of duplexes incorporating the recently described 5-nitroindole nucleoside analogue, but considerably exceed those for the 3-nitropyrrole analogue.     

Assessment of the microbiota of a mixed infection of the tongue using phenotypic and genotypic methods simultaneously and a review of the literature

We assessed the microbiota of a tongue abscess in which twelve different aerobic and anaerobic bacteria were identified using fluorescent in situ hybridisation (FISH), sequencing of the 16S rRNA gene and phenotypic methods. By applying the 16S rRNA based probes directly on the clinical material, a quick insight of the bacteria present was obtained and the species which were not cultured but present in the abscess were identified.     

The knee adduction moment measured with an instrumented force shoe in patients with knee osteoarthritis

The external knee adduction moment (KAdM) during gait is an important parameter in patients with knee osteoarthritis (OA). KAdM measurement is currently restricted to instruments only available in gait laboratories. However, ambulatory movement analysis technology, including instrumented force shoes (IFS) and inertial and magnetic measurement systems (IMMS), can measure kinetics and kinematics of human gait free of laboratory restrictions. The objective of this study was a quantitative validation of the accuracy of the KAdM in patients with knee OA, when estimated with an ambulatory-based method (AmbBM) versus a laboratory-based method (LabBM). AmbBM is employing the IFS and a linked-segment model, while LabBM is based on a force plate and optoelectronic marker system. Effects of ground reaction force (GRF), centre of pressure (CoP), and knee joint position measurement are evaluated separately. Twenty patients with knee OA were measured. The GRFs showed differences up to 0.22 N/kg, the CoPs showed differences up to 4 mm, and the medio-lateral and vertical knee position showed differences to 9 mm, between AmbBM and LabBM. The GRF caused an under-estimation in KAdM in early stance. However, this effect was counteracted by differences in CoP and joint position, resulting in a net 5% over-estimation. In midstance and late stance the accuracy of the KAdM was mainly limited by use of the linked-segment model for joint position estimation, resulting in an under-estimation (midstance 6% and late stance 22%). Further improvements are needed in the estimation of joint position from segment orientation.     

Roman impact on the landscape near castellum Fectio,The Netherlands

Castellum Fectio was one of the largest fortifications along the Limes, the northern border of the Roman Empire. The castellum, situated 5 km southeast of Utrecht, the Netherlands, was occupied from around the start of our Era to ca. a.d. 260. It was situated along a river bend of the Rhine that was cut off from the main stream during the occupation of the Roman fort. A 6 m long sediment sequence was recovered from the infill of the residual channel and pieces of Roman wall plaster, glume bases of Triticum spelta and radiocarbon dates indicate that the sediments were deposited during the period of Roman occupation. The combined palaeoecological analyses—palynological, macrobotanical, entomological and geochemical—allow a detailed reconstruction of changing environmental conditions as a consequence of the Roman occupation. The pollen record reveals a dramatic decrease in arboreal pollen, suggesting that the Romans were involved in large-scale deforestation, transforming semi-open parkland to a landscape of meadows and agricultural fields. Non-pollen palynomorphs, botanical macrofossils and insect remains support this conclusion. The recorded mycoflora shows a shift from assemblages characterised by the tree pathogen Kretzschmaria deusta to assemblages dominated by spores of fungi associated with herbaceous plants, concurrent with the decrease in arboreal pollen. The presence of masticated bran fragments of cereals, clover remains, eggs of intestinal parasites and entomological and geochemical data in the upper part of the sequence indicates that these sediments largely consist of faeces that were dumped into the former channel. Surprisingly, seeds of salt tolerant species are encountered in the sediments of this inland site, which was situated outside the influence of the sea. Horses may have brought these seeds to Fectio in their intestinal tracts after grazing in coastal meadows.     

Amino-acyl tXNA as inhibitors or amino acid donors in peptide synthesis

Xenobiotic nucleic acids (XNAs) offer tremendous potential for synthetic biology, biotechnology, and molecular medicine but their ability to mimic nucleic acids still needs to be explored. Here, to study the ability of XNA oligonucleotides to mimic tRNA, we synthesized three L-Ala-tXNAs analogs. These molecules were used in a non-ribosomal peptide synthesis involving a bacterial Fem transferase. We compared the ability of this enzyme to use amino-acyl tXNAs containing 1′,5′-anhydrohexitol (HNA), 2′-fluoro ribose (2′F-RNA) and 2′-fluoro arabinose. L-Ala-tXNA containing HNA or 2′F-RNA were substrates of the Fem enzyme. The synthesis of peptidyl-XNA and the resolution of their structures in complex with the enzyme show the impact of the XNA on protein binding. For the first time we describe functional tXNA in an in vitro assay. These results invite to test tXNA also as substitute for tRNA in translation.     

Overexpression and mass spectrometry analysis of mature human acid ceramidase

Human acid ceramidase catalyzes the last step of lysosomal sphingolipid degradation, the hydrolysis of ceramide to sphingosine and free fatty acid. Inherited deficiency of acid ceramidase activity leads to Farber disease (Farber lipogranulomatosis). In this study, we describe the overexpression and processing of recombinant human acid ceramidase in Sf21 insect cells, its purification and characterization. Infection of Sf21 cells with a recombinant baculovirus encoding acid ceramidase precursor led to a mixture of human acid ceramidase precursor and mature enzyme secreted into the medium. Acidification of the cell culture supernatant to pH 4.2-4.3 triggered the processing of the precursor and resulted in a homogeneous sample of mature human acid ceramidase. The enzyme was purified by chromatography on Concanavalin A Sepharose and Octyl Sepharose yielding 1 mg purified protein per liter of supernatant. The recombinant enzyme was deglycosylated with peptide N-glycosidase F and the main component of the released oligosaccharides was identified as GlcNAc(2)(Fuc)Man(3) by electrospray mass spectrometry. Apparently, five of the six potential N-glycosylation sites were used. Tryptic digestion of the functional recombinant enzyme and matrix-assisted laser desorption/ionization time-of-flight- and electrospray ionization-mass spectrometry analysis of the resulting peptides indicated disulfide bridges between C10-C319, C122-C271 and C367-C371.