Pilot-Scale Production of the Natural Colorant Laetiporic Acid, Its Stability and Potential Applications

Laetiporus sulphureus, a wood-decaying basidiomycete, produces yellow-orange pigments in fruiting bodies and, as was recently shown, in submerged cultivated mycelia. Out of four strains, the most potent laetiporic acid producer was identified and its yield compared in different media. The complex Moser b medium was replaced by potato dextrose broth, achieving higher yields at a lower cost. Cultivation was then scaled up from shake flask to a 7 L stirred tank bioreactor. Optimization of parameters led to increased product concentrations up to 1 g L−1, the highest yield reported so far. An in situ product recovery strategy with a biphasic system was established, increasing the yield by 19% on the shake flask scale. A crude ethanolic extract of the biomass was examined for color stability and application trials. In contrast to what has been suggested in the past, the pigment showed limited long-term stability to oxygen and light, but was stable under storage in the dark at 4 °C under nitrogen. The orange extract was successfully incorporated into different matrices like foods, cosmetics and textiles. Laetiporic acid can potentially replace petrochemical based synthetic dyes, and can thus support the development of a circular bioeconomy

Regulation of neurite dynamics in development and after injury in Drosophila melanogaster

The integration of a neuron in a functional circuit relies on the controlled formation of neurites. Neurites are the axonal and dendritic processes by which these highly specialized cells perceive and relay information. In mammalian systems a common mechanism to establish circuits is refinement of excessive projections during development. However, mechanisms of exuberant neurite refinement are largely unknown. In this work, we show that an adult specific neuronal circuit in the central visual system of Drosophila shows excessive axon branch formation and subsequent pruning during brain development. Subsequently, we analyzed the mechanisms of this developmental refinement. We find that dosage critical activation of asymmetrically distributed Epidermal Growth Factor Receptor (EGFR) by ligand released from retinal axons is necessary for branch pruning. Live imaging of the developing brain shows that EGFR signaling is required for branch growth and retraction dynamics, likely through regulation of the actin cytoskeleton. These observations establish the first Drosophila model for developmental neuronal circuit refinement in the central nervous system (CNS) and identify non-canonical localized EGFR signaling as a novel refinement mechanism. Disruption of axonal processes in the adult leads to the loss of information transfer and therefore to dysfunctions of the nervous system. To recover functionality of injured neurons, axons first need to re-initiate axon growth similar to axon formation during development. The capacity of CNS neurons to re-grow is severely reduced, in contrast to their counterparts in the peripheral nervous system.Two major causes of this inability are the inhibitory CNS environment and the loss of the intrinsic capacity to grow. Many studies focused on the inhibitory CNS environment but successful manipulation to achieve robust regeneration is still missing. Therefore increasing efforts are undertaken to uncover novel factors to enhance the intrinsic growth capacity. To this end, we conducted the first adult CNS injury screen in invertebrates using a Drosophila regeneration assay developed in our laboratory. With a targeted gain-of-function pre-screen of approximately 330 genes, we first identify 30 modulators of developmental outgrowth. Subsequently, genes with growth enhancing properties were tested in the regeneration assay. We show that genetic manipulations of three different transcription factors (Kay, Pdm2 and Sens) and a gene involved in protein turnover (faf) can enhance the regenerative response of injured axons from a small subpopulation of CNS neurons (sLNv). We prove that these factors can robustly increase the amount of brains showing a regenerative responseand significantly enhance the length these axons can grow after injury.status: publishe

A Fat-Facets-Dscam1-JNK Pathway Enhances Axonal Growth in Development and after Injury

Injury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. Here, we conducted a gain-of-function genetic screen in Drosophila to identify strong inducers of axonal growth after injury. We focus on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3′-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism

A Fat-Facets-Dscaml-JNK Pathway Enhances Axonal Growth in Development and after Injury

Injury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. Here, we conducted a gain-of-function genetic screen in Drosophila to identify strong inducers of axonal growth after injury. We focus on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3'-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism.status: publishe

A Fat-Facets-Dscam1-JNK Pathway Enhances Axonal Growth in Development and after Injury

International audienceInjury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. Here, we conducted a gain-of-function genetic screen in Drosophila to identify strong inducers of axonal growth after injury. We focus on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3′-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism

HUWE1 affects DCN axon branching.

<p>(A-C’) Axon projections of the DCN in the optic lobe, visualized via staining against mCD8-GFP. Lo = lobula, Me = Medulla. (A) Representative image of a control brain: w;UAS-mCD8-GFP/+;control^VK31/atoGal4-14a,UAS-LacZ. (A’) Magnification of the branching area in the white square shown in panel A. (B,C) Overexpression of HUWE1 in w;UAS-HUWE1^VK37/UAS-mCD8-GFP;atoGal4-14a,UAS-LacZ/+ and w;UAs-mCD8-GFP/+;UAS-HUWE1^VK31/atoGal4-14a,UAS-LacZ flies does not affect axon number in the medulla, but leads to increased axon branching at the 3^rd branching point. (B’,C’) Magnification of the branching area in the white squares shown in panels B and C. </p

The Wnt/β-catenin pathway is involved in the disturbed DCN branching.

<p>(A,B) Reduced dsh levels in w;UAS-dsh-RNAi/UAS-mCD8-GFP;atoGal4-14a,UAS-LacZ/+ and heterozygous null mutant dsh⁶/+;UAS-mCD8-GFP/+;atoGal4-14a,UAS-LacZ/+ animals also led to an increased axon branching at the 3^rd branching point. (C) DCN axon branching is normal in dsh¹;UAS-mCD8-GFP/+;atoGal4-14a,UAS-LacZ/+ males, which are only mutant in the DEP domain responsible for activation of the non-canonical pathway. (D) DCN axon branching is equally affected in dominant negative Fz2;UAS-dn-Fz2/UAS-mCD8-GFP;atoGal4-14a,UAS-LacZ/+ flies. (E) Combined expression of HUWE1 and <i>Arm</i>^<i>ACT</i> in w;UAS-Arm^ACT/UAS-mCD8-GFP;UAS-HUWE1^VK31/atoGal4-14a,UAS-LacZ flies partially rescues the branching phenotype, although the number of branches is not completely reverted to wild-type levels. (F) Expression of a constitutively active <i>Arm</i> mutant in w;UAS-Arm^ACT/UAS-mCD8-GFP;atoGal4-14a,UAS-LacZ/+ animals results in a reduced branching phenotype. (G) Model showing the association of HUWE1 with the Wnt/β-catenin pathway and its effect on axon branching and/or pruning, as evidenced by our data. (H,I) Quantitation of the axon branching levels at the 3^rd branching point of the DCNs in the medulla. We evaluated 20-25 neurons from at least 5 different brains per genotype. Error bars represent standard error of the mean (SEM) (*** p<0.001, ** p<0.01). </p