Retrospective analysis of 302 ovine dystocia cases presented to a veterinary hospital with particular attention to uterine torsion

Background Dystocia is common in sheep, and foetal causes are predominant. Among maternal causes, insufficient cervical dilatation is the most frequent problem. Uterine torsion has been considered rare by many authors. Objectives This study was conducted to investigate causes of dystocia in sheep presented for veterinary attention, and particular focus was set on the description of uterine torsion and analysis of potentially predisposing factors for this condition. Methods Clinical records of 302 sheep treated for dystocia were evaluated retrospectively. Known and proposed risk factors for uterine torsion in cattle were analysed regarding their potential importance in sheep. These included lamb birth weights, ewe age, parity, season, nutrition, breed type, litter size and husbandry. Results Maternal causes of dystocia accounted for 67.2% (203/302) of the presented cases. Of these, insufficient cervical dilatation (121/203, 59.6%) was the most frequent diagnosis. Another substantial proportion of maternal causes (60/203, 29.6%) was identified as uterine torsion. Husbandry, breed type and litter size showed significance in univariate analyses, with lower odds for meat breeds (OR 0.22; p < 0.001), twin- (OR 0.49; p = 0.020) or multiple-bearing ewes (OR 0.19; p = 0.013) and higher odds for fully housed animals (OR 17.87; p < 0.001). Year-round housing was identified as the most influential factor in a subsequent multivariate analysis. Conclusions Uterine torsion was identified as a relevant cause of dystocia in our case load. The condition is likely to be underdiagnosed in sheep, and increased farmer and veterinary awareness is necessary to ensure adequate treatment of affected animals and to prevent unnecessary suffering

Genome mining in Amycolatopsis balhimycina for ferredoxins capable of supporting cytochrome P450 enzymes involved in glycopeptide antibiotic biosynthesis

Ferredoxins are required to supply electrons to the cytochrome P450 enzymes involved in cross-linking reactions during the biosynthesis of the glycopeptide antibiotics balhimycin and vancomycin. However, the biosynthetic gene clusters for these antibiotics contain no ferredoxin- or ferredoxin reductase-like genes. In a search for potential ferredoxin partners for these P450s, here, we report an in silico analysis of the draft genome sequence of the balhimycin producer Amycolatopsis balhimycina, which revealed 11 putative Fe-S-containing ferredoxin genes. We show that two members (balFd-V and balFd-VII), produced as native-like holo-[3Fe-4S] ferredoxins in Escherichia coli, could supply electrons to the P450 OxyB (CYP165B) from both A. balhimycina and the vancomycin producer Amycolatopsis orientalis, and support in vitro turnover of peptidyl carrier protein-bound peptide substrates into monocyclic cross-linked products. These results show that ferredoxins encoded in the antibiotic-producing strain can act in a degenerate manner in supporting the catalytic functions of glycopeptide biosynthetic P450 enzymes from the same as well as heterologous gene cluster

Two small forest mires in the Hochspessart (SW Germany) as archives of landscape history and objects of nature conservation

Abstract: Pollen content, flora, and vegetation of small forest mires (< 1 ha) in the mesozoic sandstone region of the Spessart (SW Germany) are presented. According to pollen and radiocarbon data, peat formation began 600 – 700 (mire at the Birkwasser, southern Hochspessart) and 300 years ago (mire at the Ödborn, northern Hochspessart). The pollen diagrams comprise FIRBAS (1949) forest succession stages X a and b which can be correlated to the data of forest history. The present day composition of plant species and vegetation (mainly Caricetum fuscae and Caricetum rostratae) indicate oligo-/mesotrophic acid conditions. Some species, e.g. Carex canescens and Sphagnum compactum, are endangered in the Spessart-Rhön region and in Bavaria. Despite their small size the investigated mires enhance the biodiversity of the Spessart mountains on the species and habitat level and fulfil ecological functions, like the storage of water, nutrients, and harmful substances. They are also of singular and irreplaceable importance as archives of landscape history. For these reasons they have to be protected from external interference (e.g. afforestation, clearcutting) with changes in the water balance. In respect to conservation, development, and regeneration of different mire types in Central Europe, it is necessary not to neglect small forest mires as specific habitats.Zusammenfassung: Pollenführung, Flora und Vegetation zweier kleiner Waldmoore (< 1 ha) im Buntsandstein-Spessart (SW-Deutschland) werden vorgestellt. Nach pollenanalytischen und 14C-Datierungen entstanden die Moore vor 600 – 700 (Moor am Birkwasser, südlicher Hochspessart) bzw. 300 Jahren (Moor am Ödborn, nördlicher Hochspessart). Die Pollendiagramme erfassen die waldgeschichtlichen Abschnitte X a und b nach FIRBAS (1949) und ergänzen die historischen Quellen zur Wald- und Forstgeschichte des Spessarts. Die heutige Artenzusammensetzung und Vegetation der Moore (v.a. Caricetum fuscae und Caricetum rostratae) weisen auf oligo-/mesotroph-saure Standortbedingungen hin. Einige Arten, z.B. Carex canescens und Sphagnum compactum, sind in der Region Spessart-Rhön und in Bayern gefährdet bzw. potentiell gefährdet. Trotz ihrer geringen Größe tragen die untersuchten Moore zur Arten- und Biotopvielfalt der umgebenden Wälder und Forsten bei. Als Wasser-, Nährstoff- und Schadstoffspeicher übernehmen sie wichtige ökologische Funktionen im Naturhaushalt des Waldes und sind als Archive der Landschaftsgeschichte unersetzbar. Um eine Schädigung durch Eingriffe in den Wasserhaushalt zu verhindern (z.B. Aufforstungen oder Rodungen), müssen die Kleinmoore einem besonderen Schutz unterstellt werden. Vor dem Hintergrund von Erhaltung, Entwicklung, Renaturierung und Naturschutz unterschiedlicher Moortypen in Mitteleuropa sollten auch kleine Waldmoore mit ihren Sonderstrukturen nicht vernachlässigt werden.DFG, SUB Göttingen, DGMTresearc

Mire vegetation and development of Drei Seen in the central Odenwald (NW Bavaria)

Abstract: Flora, mire vegetation, actual and fossil pollen precipitation of medieval fish ponds are investigated. At present days each pond shows different stages of mire development. Small sedge communities of Caricetum fuscae and Caricetum rostratae prevail, in open waters Potamogeton natans, Juncus bulbosus and at the pond margins Carex rostrata grow. The pollen precipitation is compared with the actual vegetation cover of the mire and its 500 m radius. Soil dependent mire formation in the brook valley starts in post-Roman times. After construction of the fish ponds and increasing forest clearance the mire development continues by increasing peat formation, and in the ponds limno-minerogenic sediments accumulate fastly. Mire desiccation at the valley margins and mesotrophic fen peat formation in the remnant ponds during the last 250 years by surrounding afforestation lead to the present day status of a protected area within the Nature 2000 fauna-flora-habitat system. Proposals for nature reserve management are applied to the lake and mire biotops as well as the surrounding forests.Zusammenfassung: Flora, Moorvegetation, rezente und fossile Pollenführung der im Mittelalter angelegten Fischteiche Drei Seen wurden untersucht. Jeder der Seen weist aktuell unterschiedliche Stadien der Moorentwicklung auf. Kleinseggengesellschaften des Caricetum fuscae und Caricetum rostratae herrschen vor, im freien Wasser Potamogeton natans, Juncus bulbosus und ufernah Carex rostrata. Der Pollenniederschlag wird mit der aktuellen Vegetation im Moor und dem 500 m-Umkreis verglichen. Die soligene Moorentwicklung im Bachtal beginnt in der Nachrömerzeit. Mit Anlage der Fischteiche und den zunehmenden spätmittelalterlichen Waldrodungen geht die Moorentwicklung am Talrand in verstärkte Torfbildung über, während die limno-minerogenen Teichsedimente rasch akkumulieren. Randliche Mooraustrocknung und mesotrophe Riedtorfbildung in den Teichen führen seit den Aufforstungen der letzten 250 Jahre zum heutigen Zustand des FFH-Schutzgebietes. Vorschläge zum Biotop-Management erstrecken sich auf Gewässer, Moor und umgebende Forsten.DFG, SUB Göttingen, DGMTresearc

Protein epitope mimetics: from new antibiotics to supramolecular synthetic vaccines

Protein epitope mimetics provide powerful tools to study biomolecular recognition in many areas of chemical biology. They may also provide access to new biologically active molecules and potentially to new classes of drug and vaccine candidates. Here we highlight approaches for the design of folded, structurally defined epitope mimetics, by incorporating backbone and side chains of hot residues onto a stable constrained scaffold. Using robust synthetic methods, the structural, biological, and physical properties of epitope mimetics can be optimized, by variation of both side chain and backbone chemistry. To illustrate the potential of protein epitope mimetics in medicinal chemistry and biotechnology, we present studies in two areas of infectology; the discovery of new antibiotics targeting essential outer membrane (OM) proteins in Gram-negative bacteria and the design of supramolecular synthetic vaccines. The discovery of new antibiotics with novel mechanisms of action, in particular to combat infections caused by Gram-negative pathogens, represents a major challenge in medicinal chemistry. We were inspired by naturally occurring cationic antimicrobial peptides to design structurally related peptidomimetics and to optimize their antimicrobial properties through library synthesis and screening. Through these efforts, we could show that antimicrobial β-hairpin mimetics may have structures and properties that facilitate interactions with essential bacterial β-barrel OM proteins. One recently discovered family of antimicrobial peptidomimetics targets the β-barrel protein LptD in Pseudomonas spp. This protein plays a key role in lipopolysaccaride (LPS) transport to the cell surface during OM biogenesis. Through a highly selective interaction with LptD, the peptidomimetic blocks LPS transport, resulting in nanomolar antimicrobial activity against the important human pathogen P. aeruginosa. Epitope mimetics may also have great potential in the field of vaccinology, where structural information on complexes between neutralizing antibodies and their cognate epitopes can be taken as a starting point for B cell epitope mimetic design. In order to generate potent immune responses, an effective method of delivering epitope mimetics to relevant cells and tissues in the immune system is also required. For this, engineered synthetic nanoparticles (synthetic virus-like particles, SVLPs) prepared using supramolecular chemistry can be designed with optimal surface properties for efficient dendritic cell-mediated delivery of folded B-cell and linear T-cell epitopes, along with ligands for pattern recognition receptors, into lymphoid tissues. In this way, multivalent display of the epitope mimetics occurs over the surface of the nanoparticle, suitable for cross-linking B cell receptors. In this highly immunogenic format, strong epitope-specific humoral immune responses can be elicited that target infections caused by pathogenic microorganisms. Other potential applications of epitope mimetics in next-generation therapeutics are also discussed

Solution structure and dynamics of LptE from Pseudomonas aeruginosa

LptE is an outer membrane (OM) lipoprotein found in Gram-negative bacteria, where it forms a complex with the β-barrel lipopolysaccharide(LPS) transporter LptD. The LptD/E complex plays a key role in OM biogenesis, by translocating newly synthesized LPS molecules from the periplasm into the external leaflet of the asymmetric OM during cell growth. The LptD/E complex in Pseudomonas aeruginosa (Pa) is a target for macrocyclic β-hairpin-shaped peptidomimetic antibiotics, which inhibit the transport of LPS to the cell surface. So far, the three-dimensional structure of the Pa LptD/E complex and the mode of interaction with these antibiotics are unknown. Here, we report the solution structure of a Pa LptE derivative lacking the N-terminal lipid membrane anchor, determined by multidimensional solution nuclear magnetic resonance (NMR)spectroscopy. The structure reveals a central five-stranded β-sheet against which pack a long C-terminal and a short N-terminal α-helix, as found in homologues of LptE from other Gram-negative bacteria. One unique feature is an extended C-terminal helix in Pa LptE, which in a model of the Pa LptD/E complex appears to be long enough to contact the periplasmic domain of LptD. Chemical shift mapping experiments suggest only weak interactions occur between LptE and the oligosaccharide chains of LPS. The NMR structure of Pa LptE will be valuable for more detailed structural studies of the LptD/E complex from P. aeruginosa

Bis-chlorination of a hexapeptide-PCP conjugate by the halogenase involved in vancomycin biosynthesis

Vancomycin is an important nosocomial antibiotic containing a glycosylated, cross-linked and doubly chlorinated heptapeptide backbone. During the biosynthesis of the vancomycin aglycone, two ß-hydroxytyrosine (Bht) residues are inserted at positions-2 and -6 into the heptapeptide backbone by a non-ribosomal peptide synthetase. A single flavin-dependent chlorinase (VhaA) is responsible for chlorinating both Bht residues at some ill-defined point in the assembly process. We show here using in vitro assays that VhaA is able to introduce a chlorine atom into each aromatic ring of both Bht residues at positions-2 and -6 of a peptide carrier protein-bound hexapeptide. The results suggest that VhaA can recognize and chlorinate two quite different sites within a linear hexapeptide intermediate during vancomycin biosynthesis

Identification of genes required for resistance to peptidomimetic antibiotics by transposon sequencing

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of nosocomial infections. Due to its high intrinsic and adaptive resistance to antibiotics, infections caused by this organism are difficult to treat and new therapeutic options are urgently needed. Novel peptidomimetic antibiotics that target outer membrane (OM) proteins have shown great promise for the treatment of P. aeruginosa infections. Here, we have performed genome-wide mutant fitness profiling using transposon sequencing (Tn-Seq) to identify resistance determinants against the recently described peptidomimetics L27-11, compounds 3 and 4, as well as polymyxin B2 (PMB) and colistin (COL). We identified a set of 13 core genes that affected resistance to all tested antibiotics, many of which encode enzymes involved in the modification of the lipopolysaccharide (LPS) or control their expression. We also identified fitness determinants that are specific for antibiotics with similar structures that may indicate differences in their modes of action. These results provide new insights into resistance mechanisms against these peptide antibiotics, which will be important for future clinical development and efforts to further improve their potency

Crystal Structure of OxyC, a Cytochrome P450 Implicated in an Oxidative C–C Coupling Reaction during Vancomycin Biosynthesis

Gene inactivation studies point to the involvement of OxyC in catalyzing the last oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. Presently, the substrate and exact timing of the OxyC reaction are unknown. The substrate might be the bicyclic heptapeptide or a thioester derivative bound to a protein carrier domain. OxyC from the vancomycin producer Amycolatopsis orientalis was produced in Escherichia coli and crystallized, and its structure was determined to 1.9 Å resolution. OxyC gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. The addition of vancomycin aglycone to OxyC produced type I changes to the UV spectrum. OxyC exhibits the typical P450-fold, with the Cys ligand loop containing the signature sequence FGHGX-HXCLG and Cys-356 being the proximal axial thiolate ligand of the heme iron. The observation of a water molecule bound to the heme iron is consistent with the UV-visible spectra of OxyC indicating a low spin heme. A polyethylene glycol molecule occupying the active site might mimic the bicyclic heptapeptide substrate. Analysis of the structure of Oxy-proteins and other P450s indicates regions that might be involved in binding of the redox partner and possibly the protein carrier domain