Adult Influence on Juvenile Phenotypes by Stage-Specific Pheromone Production.

Many animal and plant species respond to population density by phenotypic plasticity. To investigate if specific age classes and/or cross-generational signaling affect density-dependent plasticity, we developed a dye-based method to differentiate co-existing nematode populations. We applied this method to Pristionchus pacificus, which develops a predatory mouth form to exploit alternative resources and kill competitors in response to high population densities. Remarkably, adult, but not juvenile, crowding induces the predatory morph in other juveniles. High-performance liquid chromatography-mass spectrometry of secreted metabolites combined with genetic mutants traced this result to the production of stage-specific pheromones. In particular, the P. pacificus-specific di-ascaroside#1 that induces the predatory morph is induced in the last juvenile stage and young adults, even though mouth forms are no longer plastic in adults. Cross-generational signaling between adults and juveniles may serve as an indication of rapidly increasing population size, arguing that age classes are an important component of phenotypic plasticity.

Beyond the Modification Degree: Impact of Raw Material on Physicochemical Properties of Gelatin Type A and Type B Methacryloyls.

Gelatin methacryloyl (acetyl) (GM(A)) is increasingly investigated for various applications in life sciences and medicine, for example, drug release or tissue engineering. Gelatin type A and type B are utilized for GA M(A) and GB M(A) preparation, but the impact of gelatin raw material on modification reaction and resulting polymer properties is rather unknown so far. Therefore, the degrees of modification (DMA) and physicochemical properties of five GA M(A) and GB M(A) derivatives are compared: The degrees of methacryloylation (0.32-0.98 mmol g-1 ) are indistinguishable for GA M(A) and GB M(A) as are the sol-gel temperatures. Isoelectric points, solution viscosities, and hydrodynamic radii which are distinct for GA and GB, converge with increasing DMA. Interestingly, differences are measured for the storage moduli and equilibrium degrees of swelling of respective GA and GB derivative-based hydrogels, in spite of their comparable DMA. This underlines the importance of GM(A) characterization beyond the modification degree.

Photoinduced Cleavage and Hydrolysis of o-Nitrobenzyl Linker and Covalent Linker Immobilization in Gelatin Methacryloyl Hydrogels.

Light-induced release systems can be triggered remotely and are of interest for many controlled release applications due to the possibility for spatio-temporal release control. In this study a biotin-functionalized photocleavable macromer is incorporated with an o-nitrobenzyl moiety into gelatin methacryloyl(-acetyl) hydrogels via radical cross-linking. Stronger immobilization of streptavidin-coupled horseradish peroxidase occurs in linker-functionalized hydrogels compared to pure gelatin methacryloyl(-acetyl) hydrogels, and a controlled release of the streptavidin conjugate upon UV-irradiation is possible. Liquid chromatography coupled to mass spectrometry (LC-MS) analysis of aqueous linker solutions allows the identification of the main cleavage products and the cleavage kinetics. Thus, it is shown that a significant hydrolysis of the linker occurs at 37 °C. Nevertheless the system reported here is a promising controlled release scaffold for proteins and application in tissue engineering, if background releases of the immobilized drug are tolerable.

Linking Genomic and Metabolomic Natural Variation Uncovers Nematode Pheromone Biosynthesis.

In the nematodes Caenorhabditis elegans and Pristionchus pacificus, a modular library of small molecules control behavior, lifespan, and development. However, little is known about the final steps of their biosynthesis, in which diverse building blocks from primary metabolism are attached to glycosides of the dideoxysugar ascarylose, the ascarosides. We combine metabolomic analysis of natural isolates of P. pacificus with genome-wide association mapping to identify a putative carboxylesterase, Ppa-uar-1, that is required for attachment of a pyrimidine-derived moiety in the biosynthesis of ubas#1, a major dauer pheromone component. Comparative metabolomic analysis of wild-type and Ppa-uar-1 mutants showed that Ppa-uar-1 is required specifically for the biosynthesis of ubas#1 and related metabolites. Heterologous expression of Ppa-UAR-1 in C. elegans yielded a non-endogenous ascaroside, whose structure confirmed that Ppa-uar-1 is involved in modification of a specific position in ascarosides. Our study demonstrates the utility of natural variation-based approaches for uncovering biosynthetic pathways.

Quantification of Substitution of Gelatin Methacryloyl: Best Practice and Current Pitfalls.

Cross-linkable gelatin methacryloyl (GM) is widely used for the generation of artificial extracellular matrix (ECM) in tissue engineering. However, the quantification of modified groups in GM is still an unsolved issue, although this is the key factor for tailoring the physicochemical material properties. In this contribution, 1H-13C-HSQC NMR spectra are used to gain detailed structural information on GMs and of 2-fold modified gelatin containing methacryloyl and acetyl groups (GMAs). Distinctive identification of methacrylate, methacrylamide, and acetyl groups present in GMs and GMAs revealed an overlap of methacrylamide and modified hydroxyproline signals in the 1H NMR spectrum. Considering this, we suggest a method to quantify methacrylate and methacrylamide groups in GMs precisely based on simple 1H NMR spectroscopy with an internal standard. Quantification of acetylation in GMAs is also possible, yet, 2D NMR spectra are necessary. The described methods allow direct quantification of modified groups in gelatin derivatives, making them superior to other, indirect methods known so far.

Functional Conservation and Divergence of daf-22 Paralogs in Pristionchus pacificus Dauer Development.

Small-molecule signaling in nematode dauer formation has emerged as a major model to study chemical communication in development and evolution. Developmental arrest as nonfeeding and stress-resistant dauer larvae represents the major survival and dispersal strategy. Detailed studies in Caenorhabditis elegans and Pristionchus pacificus revealed that small-molecule communication changes rapidly in evolution resulting in extreme structural diversity of small-molecule compounds. In C. elegans, a blend of ascarosides constitutes the dauer pheromone, whereas the P. pacificus dauer pheromone includes additional paratosides and integrates building blocks from diverse primary metabolic pathways. Despite this complexity of small-molecule structures and functions, little is known about the biosynthesis of small molecules in nematodes outside C. elegans Here, we show that the genes encoding enzymes of the peroxisomal ß-oxidation pathway involved in small-molecule biosynthesis evolve rapidly, including gene duplications and domain switching. The thiolase daf-22, the most downstream factor in C. elegans peroxisomal ß-oxidation, has duplicated in P. pacificus, resulting in Ppa-daf-22.1, which still contains the sterol-carrier-protein (SCP) domain that was lost in C. elegans daf-22, and Ppa-daf-22.2. Using the CRISPR/Cas9 system, we induced mutations in both P. pacificus daf-22 genes and identified an unexpected complexity of functional conservation and divergence. Under well-fed conditions, ascaroside biosynthesis proceeds exclusively via Ppa-daf-22.1 In contrast, starvation conditions induce Ppa-daf-22.2 activity, resulting in the production of a specific subset of ascarosides. Gene expression studies indicate a reciprocal up-regulation of both Ppa-daf-22 genes, which is, however, independent of starvation. Thus, our study reveals an unexpected functional complexity of dauer development and evolution.