Using latent effects to determine the ecological importance of dissolved organic matter to marine invertebrates

The uptake and utilization of dissolved organic matter (DOM)by marine invertebrates is a field that has received significantattention over the past 100 years. Although it is well establishedthat DOM is taken up by marine invertebrates, the extent towhich it contributes to an animal's survival, growth, and reproduction(that is, the ecological benefits) remains largely unknown.Previous work seeking to demonstrate the putative ecologicalbenefits of DOM uptake have examined them within a single lifestage of an animal. Moreover, most of the benefits are demonstratedthrough indirect approaches by examining (1) mass balance, or(2) making comparisons of oxyenthalpic conversions of transportrates to metabolic rate as judged by oxygen consumption. Wesuggest that directly examining delayed metamorphosis or thelatent effects associated with nutritional stress of larvaeis a better model for investigating the ecological importanceof DOM to marine invertebrates. We also provide direct evidencethat availability of DOM enhances survival and growth of thebryozoan Bugula neritina. That DOM offsets latent effects inB. neritina suggests that the underlying mechanisms are at leastin part energetic.     

Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM₆), octasaccharide (HM₈), and decasaccharide (HM₁₀), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM₈ and HM₁₀ are significantly more potent than HM₆ in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM₈ relative to HM₆ in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM₈ to bind and dimerize FGF2. Notably FGF2·HM₈ exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.     

Comparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA-based HLA crossmatch procedure

The detection of donor-specific anti-HLA antibodies by standard procedures such as complement-dependent cytotoxicity assay (CDC) or flow cytometric (FACS) analysis is limited by its low sensitivity and the quality of the donor cells. Therefore, an ELISA-based technique was employed using solid phase-immobilized monoclonal antibodies to capture HLA class I or class II molecules of the donor, respectively. In this HLA class I and class II antibody monitoring system (AMS) the donor-specific anti-HLA antibodies from the sera of recipients bind to the HLA molecules of the donor which have been immobilized by monoclonal antibodies (mAb) recognizing non-polymorphic epitopes. Upon binding of donor-specific anti-HLA antibodies they are recognized by secondary enzyme-conjugated anti-human immunoglobulin (Ig) antibodies. A newly established modification of the standard protocol allows the differentiation between bound antibodies of the IgG and IgM isotype. Furthermore, this assay was adapted for investigating small amounts of solid tissue of donors from whom no other cells (e.g. from blood) were available. We here provide an overview of the classical crossmatch methods with their advantages and limits. In addition, the design of the novel AMS-ELISA is described in terms of quality and sensitivity of the approach using exemplary cases of different application. The selected cases show that the AMS-ELISA represents a valuable tool for the post-transplantation monitoring of donor-specific anti-HLA antibodies during reaction crisis, after transfusion reactions and in particular cases of tissue transplantations lacking single cells.     

Uniform tissues engineered by seeding and culturing cells in 3D scaffolds under perfusion at defined oxygen tensions

In this work, we assessed whether culture of uniformly seeded chondrocytes under direct perfusion, which supplies the cells with normoxic oxygen levels, can maintain a uniform distribution of viable cells throughout porous scaffolds several milimeters in thickness, and support the development of uniform tissue grafts. An integrated bioreactor system was first developed to streamline the steps of perfusion cell seeding of porous scaffolds and perfusion culture of the cell-seeded scaffolds. Oxygen tensions in perfused constructs were monitored by in-line oxygen sensors incorporated at the construct inlet and outlet. Adult human articular chondrocytes were perfusion-seeded into 4.5 mm thick foam scaffolds at a rate of 1 mm/s. Cell-seeded foams were then either cultured statically in dishes or further cultured under perfusion at a rate of 100 microm/s for 2 weeks. Following perfusion seeding, viable cells were uniformly distributed throughout the foams. Constructs subsequently cultured statically were highly heterogeneous, with cells and matrix concentrated at the construct periphery. In contrast, constructs cultured under perfusion were highly homogeneous, with uniform distributions of cells and matrix. Oxygen tensions of the perfused medium were maintained near normoxic levels (inlet congruent with 20%, outlet > 15%) at all times of culture. We have demonstrated that perfusion culture of cells seeded uniformly within porous scaffolds, at a flow rate maintaining a homogeneous oxygen supply, supports the development of uniform engineering tissue grafts of clinically relevant thicknesses.     

The Cassian patch reefs (lower Carnian,southern Alps)

The fauna of the upper Cassian Formation is composed mainly of reef-building and reef-dwelling organisms which occur as reeeposited material in basinal sediments, but have not been found as original reef bodies. Such bodies have now been discovered in the uppermost Cassian Formation of the central Dolomites from the Sella Group in the west to the Monti Cadini in the east. Generally they are small-scale patch reefs, not exceeding a few metres in thickness and lateral extent, which are intercalated in well-bedded detrital and micritic limestones. locally, larger biostromes spread out from the margins of the Cassian Dolomite buildups. Four types of faunal communities have been encountered in these reefs:

1. The thrombolite-calcareous algae community, composed of small patchy cryptalgal structures binding poorly sorted debris and associated with other Cyanophyta, sessile formainifera and scattered calcareous sponges and corals. This type is the most common within the calcareous and marly-tuffaceous facies of the Cassian Formation.
2. The calcareous sponge-coral community, composed mostly of calcareous sponges (stromatoporoids, some pharetronids) and, to a lesser extent, colonial corals and thrombolites. This community corre-sponds well to the Cassian reef fauna, best known from erratic blocks at Alpe di Specie, but has been found in situ only at one locality.
3. The Spongiomorpha-Solenopora community, associated with scattered calcareous sponges and colonial corals, forming a thin biostrome at one locality.
4. Coral communities, composed predominantly of colonial Scleractinia; found only in small or stratigraphically illdefined outcrops and in erratic blocks.

The Cassian patch reefs and biostromes mark the end of a basinal evolution which began in the Lower Ladinian, and the onset of newly expanding carbonate buildups of Cassian Dolomite. These buildups and the sponge-coral patch reefs might have been the source for the allochthonous reef fauna of the Cassian Formation which interfingers with both shallow water environments.     

Computational evaluation of oxygen and shear stress distributions in 3D perfusion culture systems: macro-scale and micro-structured models

We present a combined macro-scale/micro-scale computational approach to quantify oxygen transport and flow-mediated shear stress to human chondrocytes cultured in three-dimensional scaffolds in a perfusion bioreactor system. A macro-scale model was developed to assess the influence of the bioreactor design and to identify the proper boundary conditions for the micro-scale model. The micro-scale model based on a micro-computed tomography (microCT) reconstruction of a poly(ethylene glycol terephthalate)/poly(butylene terephthalate) (PEGT/PBT) foam scaffold, was developed to assess the influence of the scaffold micro-architecture on local shear stress and oxygen levels within the scaffold pores. Experiments were performed to derive specific oxygen consumption rates for constructs perfused under flow rates of 0.3 and 0.03 ml min(-1). While macro-scale and micro-scale models predicted similar average oxygen levels at different depths within the scaffold, microCT models revealed small local oxygen variations within the scaffold micro-architecture. The combined macro-scale/micro-scale approach indicated that 0.3 ml min(-1), which subjected 95% of the cells to less than 6.3 mPa shear, would maintain the oxygen supply throughout the scaffold above anoxic levels (>1%), with 99.5% of the scaffold supplied with 8-2% O(2). Alternatively, at 0.03 ml min(-1), the macro-scale model predicted 6% of the cells would be supplied with 0.5-1% O(2), although this region of cells was confined to the periphery of the scaffold. Together with local variations predicted by the micro-scale model, the simulations underline that in the current model system, reducing the flow below 0.03 ml min(-1) would likely have dire consequences on cell viability to pronounced regions within the engineered construct. The presented approach provides a sensitive tool to aid efficient bioreactor optimization and scaffold design.     

Thermodynamic characterization of allosteric glycogen phosphorylase inhibitors

Glycogen phosphorylase (GP) is a validated target for the treatment of type 2 diabetes. Here we describe highly potent GP inhibitors, AVE5688, AVE2865, and AVE9423. The first two compounds are optimized members of the acyl urea series. The latter represents a novel quinolone class of GP inhibitors, which is introduced in this study. In the enzyme assay, both inhibitor types compete with the physiological activator AMP and act synergistically with glucose. Isothermal titration calorimetry (ITC) shows that the compounds strongly bind to nonphosphorylated, inactive GP (GPb). Binding to phosphorylated, active GP (GPa) is substantially weaker, and the thermodynamic profile reflects a coupled transition to the inactive (tense) conformation. Crystal structures confirm that the three inhibitors bind to the AMP site of tense state GP. These data provide the first direct evidence that acyl urea and quinolone compounds are allosteric inhibitors that selectively bind to and stabilize the inactive conformation of the enzyme. Furthermore, ITC reveals markedly different thermodynamic contributions to inhibitor potency that can be related to the binding modes observed in the cocrystal structures. For AVE5688, which occupies only the lower part of the bifurcated AMP site, binding to GPb (Kd = 170 nM) is exclusively enthalpic (Delta H = -9.0 kcal/mol, TDelta S = 0.3 kcal/mol). The inhibitors AVE2865 (Kd = 9 nM, Delta H = -6.8 kcal/mol, TDelta S = 4.2 kcal/mol) and AVE9423 (Kd = 24 nM, Delta H = -5.9 kcal/mol, TDelta S = 4.6 kcal/mol) fully exploit the volume of the binding pocket. Their pronounced binding entropy can be attributed to the extensive displacement of solvent molecules as well as to ionic interactions with the phosphate recognition site.     

Genetic paternity analyses in Little Owls (Athene noctua): does the high rate of paternal care select against extra-pair young?

Summary Previous studies have shown that extra-pair fertilizations are much less frequent in Non-Passeriformes, especially in raptors, than in Passeriformes. Low breeding densities, high breeding synchrony and high rates of paternal effort have been discussed as possible causes of these low extra-pair fertilization rates. Using DNA fingerprinting, we studied the mating system of Little Owls (Athene noctua) in a population of relatively high breeding density and comparatively low breeding synchrony. We found no cases of extra-pair fertilization among 53 nestlings of 16 breeding pairs. We conclude that paternal effort is probably the most important factor in preventing extra-pair fertilizations in Little Owls.

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Genetische Vaterschaftsanalysen bei Steinkäuzen (Athene noctua): Beeinflußt der hohe elterliche Aufwand der Männchen das Auftreten von Vaterschaften außerhalb des Paarbundes?

Zusammenfassung Zahlreiche Untersuchungen haben gezeigt, dass Befruchtungen außerhalb des Paarbundes bei Nicht-Singvogelarten wesentlich seltener vorkommen als bei Singvögeln. Dies gilt insbesondere auch für Greifvögel. Als Ursache für das seltene Auftreten von Befruchtungen außerhalb des Paarbundes in dieser Gruppe werden niedrige Brutpaardichten, eine hohe Brutsynchronisation und ein hoher elterlicher Aufwand auf Seiten der Männchen diskutiert. In der vorliegenden Studie haben wir das Paarungssystem des Steinkauzes (Athene noctua) in einer Population im Kreis Viersen (Niederrhein) mit Hilfe des DNA-Fingerprinting untersucht. Diese Population wies eine relativ hohe Brutpaardichte und eine vergleichsweise niedrige Brutsynchronisation auf. Bei der Analyse von 16 Bruten, die insgesamt 53 Nestlinge enthielten, konnte kein einziger Fall einer Befruchtung außerhalb des Paarbundes nachgewiesen werden. Dies führt uns zu dem Schluss, dass der wichtigste Faktor für die genetische Monogamie — zumindest beim Steinkauz — das hohe Maß des väterlichen Aufwandes bei der Brutversorgung ist.