Maternal homozygosity for three independent mutant hecate alleles results in embryos with reduced expression of dorsal organizer genes and defects in the formation of dorsoanterior structures. A positional cloning approach identified all hecate mutations as stop codons affecting the same gene, revealing that hecate encodes the Glutamate receptor interacting protein 2a (Grip2a), a protein containing multiple PDZ domains known to interact with membrane-associated factors including components of the Wnt signaling pathway. We find that grip2a mRNA is localized to the vegetal pole of the oocyte and early embryo, and that during egg activation this mRNA shifts to an off-center vegetal position corresponding to the previously proposed teleost cortical rotation. hecate mutants show defects in the alignment and bundling of microtubules at the vegetal cortex, which result in defects in the asymmetric movement of wnt8a mRNA as well as anchoring of the kinesin-associated cargo adaptor Syntabulin. We also find that, although short-range shifts in vegetal signals are affected in hecate mutant embryos, these mutants exhibit normal long-range, animally directed translocation of cortically injected dorsal beads that occurs in lateral regions of the yolk cortex. Furthermore, we show that such animally-directed movement along the lateral cortex is not restricted to a single arc corresponding to the prospective dorsal region, but occur in multiple meridional arcs even in opposite regions of the embryo. Together, our results reveal a role for Grip2a function in the reorganization and bundling of microtubules at the vegetal cortex to mediate a symmetry-breaking short-range shift corresponding to the teleost cortical rotation. The slight asymmetry achieved by this directed process is subsequently amplified by a general cortical animally-directed transport mechanism that is neither dependent on hecate function nor restricted to the prospective dorsal axis.

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In this study we investigate the hydrolysis of C3 to C3(H 2 O) and its ability to initiate activation via the alternative pathway (AP) of the complement system. The internal thioester bond within C3 is hydrolyzed by water in plasma because of its inherent lability. This results in the formation of non-proteolytically activated C3(H 2 O) which is believed have C3b-like properties and be able to form an active initial fluid phase C3 convertase together with Factor B (FB). The generation of C3(H 2 O) occurs at a low but constant rate in blood, but the formation can be greatly accelerated by the interaction with various surfaces or nucleophilic and chaotropic agents. In order to more specifically elucidate the relevance of the C3(H 2 O) for AP activation, formation was induced in solution by repeated freeze/thawing, methylamine or KCSN treatment and named C3(x) where the x can be any of the reactive nucleophilic or chaotropic agents. Isolation and characterization of C3(x) showed that it exists in several forms with varying attributes, where some have more C3b-like properties and can be cleaved by Factor I in the presence of Factor H. However, in common for all these variants is that they are less active partners in initial formation of the AP convertase compared with the corresponding activity of C3b. These observations support the idea that formation of C3(x) in the fluid phase is not a strong initiator of the AP. It is rather likely that the AP mainly acts as an amplification mechanism of complement activation that is triggered by deposition of target-bound C3b molecules generated by other means.

Studying innate immunity in humans is crucial for understanding its role in the pathophysiology of systemic inflammation, particularly in the complex setting of sepsis. Therefore, we standardized a step-by-step process from the venipuncture to the transfer in a human model system, while closely monitoring the inflammatory response for up to three hours. We designed an animal-free, human whole blood sepsis model using a commercially available, simple to use, tubing system. First, we analyzed routine clinical parameters, including cell count and blood gas analysis. Second, we demonstrated that extracellular activation markers (e.g., CD11b and CD62l) as well as intracellular metabolic (intracellular pH) and functional (generation of radical oxygen species) features remained stable after incubation in the whole blood model. Third, we mimicked systemic inflammation during early sepsis by exposure of whole blood to pathogen-associated molecular patterns. Stimulation with lipopolysaccharide revealed the capability of the model system to evoke a sepsis-like inflammatory phenotype of innate immunity. In summary, the presented model serves as a convenient, economic, and reliable platform to study innate immunity in human whole blood, which may yield clinically important insights.

Polyethylene glycol (PEG) is frequently used for liposome surface modification. However, as PEGylated liposomes are cleared rapidly from circulation upon repeated injections, substitutes of PEG are being sought. We focused...