Dityrosine Cross-linking Research Articles

Bioengineered Silk Fibroin Hydrogel Reinforced with Collagen-Like Protein Chimeras for Improved Wound Healing.

The study investigates the potentials of the rapid crosslinking hydrogel concoction comprising of natural silk fibroin (SF) and recombinant tailorable collagen-like protein with binding domains for wound repair. The formation of dityrosine crosslinks between the tyrosine moieties augments the formation of stable hydrogels, in the presence of the cytocompatible photo-initiator riboflavin and visible light. This uniquely engineered PASCH (Photo-activated silk fibroin and tailor-made collagen-like protein hydrogel) confers the key advantage of improved biological properties over the control hydrogels comprising only of SF. The physico-chemical characterization of the hydrogels with respect to crosslinking, modulus, and thermal stability delineates the ascendancy of PASCH 7:3 over other combinations. Furthermore, the hybrid protein hydrogel proves to be a favorable cellular matrix as it enhances cell adhesion, elongation, growth, and proliferation in vitro. Time-lapse microscopy studies reveal an enhanced wound closure in human endothelial cell monolayer (EA.hy926), while the gene expression studies portray the dynamic interplay of cytokines and growth factors in the wound milieu facilitating the repair and regeneration of cells, sculpted by the proteins. The results demonstrate the improved physical and biological properties of fabricated PASCH, depicting their synergism, and implying their competency for use in tissue engineering applications.

Promoting ovalbumin amyloid fibrils formation by cold plasma treatment and improving its emulsifying properties

The potential of cold plasma (CP) treatment to promote the formation of amyloid fibrils (AFs) of ovalbumin (OVA) was evaluated relative to acidic heat fibrillation condition (pH = 2, 85 °C). Results indicated CP exhibited significant potential for promoting the formation of AFs of OVA, as evidenced by higher Thioflavin T (ThT) fluorescence intensity of AFs for CP-treated OVA (POVA) during the fibrillation process compared to AFs for native OVA under acidic heating conditions (NOVA). Long, curved and worm-like fibrils of CP-treated OVA with 2 min (POVA-2), which were thicker (3 nm in height) and longer (majority length ranging from 300 nm to 400 nm), were observed after 8 h of fibrillation, while irregular, short, worm-like fibrils of NOVA were detected. Unlike NOVA, which was only hydrolyzed into small peptides, the backbone of POVA-2 was cleaved into small peptide fragments, accompanied by the generation of dityrosine cross-linked aggregation/oligomer during fibrillation process, as evidenced by SDS-PAGE and dityrosine analysis. The result of hydrophobic, sulfhydryl and disulfide bonds, and dityrosine analysis showed hydrophobic interaction and dityrosine cross-links could be the main force driving the assembly and stacking of cross-β structures, leading to the formation of organized fibrillar structures for POVA-2, while only hydrophobic interaction was involved for NOVA. Additionally, analyses of emulsifying ability (EAI) and stability (ESI) of NOVA and POVA-2 displayed that both EAI and ESI of them were significantly improved, with POVA-2 exhibiting superior EAI compared to NOVA. Therefore, this study demonstrated CP is a promising technique to promote the generation of protein amyloid fibrils in a more efficient manner.

Knockdown of Sec16 causes early lethality and defective deposition of the protein Rp30 in the eggshell of the vector Rhodnius prolixus.

In nearly every species of insect, embryonic development takes place outside of the mother's body and is entirely dependent on the elements that the mother had previously stored within the eggs. It is well known that the follicle cells (FCs) synthesize the eggshell (chorion) components during the process of choriogenesis, the final step of oogenesis before fertilization. These cells have developed a specialization in the massive production of chorion proteins, which are essential for the protection and survival of the embryo. Here, we investigate the function of Sec16, a protein crucial for the endoplasmic reticulum (ER) to Golgi traffic, in the oocyte development in the insect Rhodnius prolixus. We discovered that Sec16 is strongly expressed in vitellogenic females' ovaries, particularly in the choriogenic oocyte and it is mainly associated with the FCs. Silencing of Sec16 by RNAi caused a sharp decline in oviposition rates, F1 viability, and longevity in adult females. In the FCs, genes involved in the unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and autophagy were massively upregulated, whereas the mRNAs of Rp30 and Rp45-which code for the two major chorion proteins - were downregulated as a result of Sec16 silencing, indicating general proteostasis disturbance. As a result, the outer surface ultrastructure of Sec16-silenced chorions was altered, with decreased thickness, dityrosine crosslinking, sulfur signals, and lower amounts of the chorion protein Rp30. These findings collectively demonstrate the critical role Sec16 plays in the proper functioning of the FCs, which impacts the synthesis and deposition of particular components of the chorion as well as the overall reproduction of this vector.

Photocrosslinkable triple helical protein with enhanced higher-order formation for biomaterial applications.

Bacterial collagen, produced via recombinant DNA methods, offers advantages including consistent purity, customizable properties, and reduced allergy potential compared to animal-derived collagen. Its controlled production environment enables tailored features, making it more sustainable, non-pathogenic, and compatible with diverse applications in medicine, cosmetics, and other industries. Research has focused on the engineering of collagen-like proteins to improve their structure and function. The study explores the impact of introducing tyrosine, an amino acid known for its rolein fibril formation across diverse proteins, into a newly designed bacterial collagen-like protein (Scl2), specifically examining its effect on self-assembly and fibril formation. Biophysical analyses reveal that the introduction of tyrosine residues didn't compromise the protein's structural stability but rather promoted self-assembly, resulting in the creation of nanofibrils-a phenomenon absent in the native Scl2 protein. Additionally, stable hydrogels are formed when the engineered protein undergoes di-tyrosine crosslinking under light exposure. The hydrogels, shown to support cell viability, also facilitate accelerated wound healing in mouse fibroblast (NIH/3T3) cells. These outcomes demonstrate that the targeted inclusion of functional residues in collagen-like proteins enhances fibril formation and facilitates the generation of robust hydrogels using riboflavin chemistry, presenting promising paths for research in tissue engineering and regenerative medicine.

Engineered dityrosine-bonding of the RSV prefusion F protein imparts stability and potency advantages

Viral fusion proteins facilitate cellular infection by fusing viral and cellular membranes, which involves dramatic transitions from their pre- to postfusion conformations. These proteins are among the most protective viral immunogens, but they are metastable which often makes them intractable as subunit vaccine targets. Adapting a natural enzymatic reaction, we harness the structural rigidity that targeted dityrosine crosslinks impart to covalently stabilize fusion proteins in their native conformations. We show that the prefusion conformation of respiratory syncytial virus fusion protein can be stabilized with two engineered dityrosine crosslinks (DT-preF), markedly improving its stability and shelf-life. Furthermore, it has 11X greater potency as compared with the DS-Cav1 stabilized prefusion F protein in immunogenicity studies and overcomes immunosenescence in mice with simply a high-dose formulation on alum.

Oligomerization and tyrosine nitration enhance the allergenic potential of the birch and grass pollen allergens Bet v 1 and Phl p 5.

Protein modifications such as oligomerization and tyrosine nitration alter the immune response to allergens and may contribute to the increasing prevalence of allergic diseases. In this mini-review, we summarize and discuss relevant findings for the major birch and grass pollen allergens Bet v 1 and Phl p 5 modified with tetranitromethane (laboratory studies), peroxynitrite (physiological processes), and ozone and nitrogen dioxide (environmental conditions). We focus on tyrosine nitration and the formation of protein dimers and higher oligomers via dityrosine cross-linking and the immunological effects studied.

Deficiency of Brummer lipase disturbs lipid mobilization and locomotion, and impairs reproduction due to defects in the eggshell ultrastructure in the insect vector Rhodnius prolixus

Rhodnius prolixus is a hematophagous insect, which feeds on large and infrequent blood meals, and is a vector of trypanosomatids that cause Chagas disease. After feeding, lipids derived from blood meal are stored in the fat body as triacylglycerol, which is recruited under conditions of energy demand by lipolysis, where the first step is catalyzed by the Brummer lipase (Bmm), whose orthologue in mammals is the adipose triglyceride lipase (ATGL). Here, we investigated the roles of Bmm in adult Rhodnius prolixus under starvation, and after feeding. Its gene (RhoprBmm) was expressed in all the analyzed insect organs, and its transcript levels in the fat body were not altered by nutritional status. RNAi-mediated knockdown of RhoprBmm caused triacylglycerol retention in the fat body during starvation, resulting in larger lipid droplets and lower ATP levels compared to control females. The silenced females showed decreased flight capacity and locomotor activity. When RhoprBmm knockdown occurred before the blood meal and the insects were fed, the females laid fewer eggs, which collapsed and showed low hatching rates. Their hemolymph had reduced diacylglycerol content and vitellogenin concentration. The chorion (eggshell) of their eggs had no difference in hydrocarbon amounts or in dityrosine crosslinking levels compared to control eggs. However, it showed ultrastructural defects. These results demonstrated that Bmm activity is important not only to guarantee lipid mobilization to maintain energy homeostasis during starvation, but also for the production of viable eggs after a blood meal, by somehow contributing to the right formation of the egg chorion.

Flavin mononucleotide in visible light photoinitiating systems for multiple-photocrosslinking and photoencapsulation strategies

Visible light-induced photocrosslinking techniques have attracted significant attention for their flexibility, controllability, safety, and energy conservation, especially in tissue engineering and biofabrication, compared to UV photocrosslinking. Despite these advantages, current photoinitiators are constrained by various challenges, including inadequate photoinitiation efficiency, low biocompatibility, poor water solubility, and limited compatibility with diverse crosslinking systems. Here, a water-soluble derivative of riboflavin, flavin mononucleotide (FMN−), was used to assess its potential as an initiator in multiple-photocrosslinking systems, including radical photopolymerization, dityrosine, and ditryptophan coupling crosslinking, under blue light irradiation. Blue light irradiation facilitated an efficient electron transfer reaction between FMN− and persulfate, owing to their suitable spectral compatibility and photoactivity. The resulting oxidizing free radicals and excited triplet state of FMN− served as initiating active species for the multiple-photocrosslinking reactions. The combination of FMN− and potassium persulfate (KPS) exhibited exceptional photoinitiation efficiency for various biomaterials, including silk fibroin, gelatin, poly(ethylene glycol) diacrylate, and carboxymethyl cellulose modified with amino acids. Furthermore, the cytocompatibility of the FMN−/KPS photoinitiator was demonstrated by the survival rates of 3T3-LI fibroblasts encapsulated in it, which exceeded 95 % when compared to a commercial initiator. Statement of significanceBy introducing persulfate, the photoinitiation efficiency of flavin mononucleotide was significantly improved. The application scenarios of flavin mononucleotide and persulfate combinations were also greatly extended, including radical photopolymerization, dityrosine, diphenylalanine, and ditryptophan coupling crosslinking. Among them, the coupling crosslinking of amino acids (di-phenylalanine, and di-tryptophan) modified carboxymethyl cellulose, to our knowledge, was first reported. The excellent cytocompatibility of cell encapsulation further proved that the combinations of flavin mononucleotide and persulfate have great potential in tissue engineering.

Peroxidase activity of rice (Oryza sativa) hemoglobin: distinct role of tyrosines 112 and 151.

Five non-symbiotic hemoglobins (nsHb) have been identified in rice (Oryza sativa). Previous studies have shown that stress conditions can induce their overexpression, but the role of those globins is still unclear. To better understand the functions of nsHb, the reactivity of rice Hb1 toward hydrogen peroxide (H2O2) has been studied in vitro. Our results show that recombinant rice Hb1 dimerizes through dityrosine cross-links in the presence of H2O2. By site-directed mutagenesis, we suggest that tyrosine 112 located in the FG loop is involved in this dimerization. Interestingly, this residue is not conserved in the sequence of the five rice non-symbiotic hemoglobins. Stopped-flow spectrophotometric experiments have been performed to measure the catalytic constants of rice Hb and its variants using the oxidation of guaiacol. We have shown that Tyrosine112 is a residue that enhances the peroxidase activity of rice Hb1, since its replacement by an alananine leads to a decrease of guaiacol oxidation. In contrast, tyrosine 151, a conserved residue which is buried inside the heme pocket, reduces the protein reactivity. Indeed, the variant Tyr151Ala exhibits a higher peroxidase activity than the wild type. Interestingly, this residue affects the heme coordination and the replacement of the tyrosine by an alanine leads to the loss of the distal ligand. Therefore, even if the amino acid at position 151 does not participate to the formation of the dimer, this residue modulates the peroxidase activity and plays a role in the hexacoordinated state of the heme.

Large-Scale Qualitative and Quantitative Assessment of Dityrosine Crosslinking Omics in Response to Endogenous and Exogenous Hydrogen Peroxide in Escherichia coli.

Excessive hydrogen peroxide causes oxidative stress in cells. The oxidation of two tyrosine residues in proteins can generate o,o'-dityrosine, a putative biomarker for protein oxidation, which plays critical roles in a variety of organisms. Thus far, few studies have investigated dityrosine crosslinking under endogenous or exogenous oxidative conditions at the proteome level, and its physiological function remains largely unknown. In this study, to investigate qualitative and quantitative dityrosine crosslinking, two mutant Escherichia coli strains and one mutant strain supplemented with H2O2 were used as models for endogenous and exogenous oxidative stress, respectively. By integrating high-resolution liquid chromatography-mass spectrometry and bioinformatic analysis, we created the largest dityrosine crosslinking dataset in E. coli to date, identifying 71 dityrosine crosslinks and 410 dityrosine loop links on 352 proteins. The dityrosine-linked proteins are mainly involved in taurine and hypotaurine metabolism, citrate cycle, glyoxylate, dicarboxylate metabolism, carbon metabolism, etc., suggesting that dityrosine crosslinking may play a critical role in regulating the metabolic pathways in response to oxidative stress. In conclusion, we have reported the most comprehensive dityrosine crosslinking in E. coli for the first time, which is of great significance in revealing its function in oxidative stress.

Dityrosine cross-linking and its potential roles in Alzheimer's disease.

Oxidative stress is a significant source of damage that accumulates during aging and contributes to Alzheimer's disease (AD) pathogenesis. Oxidation of proteins can give rise to covalent links between adjacent tyrosines known as dityrosine (DiY) cross-linking, amongst other modifications, and this observation suggests that DiY could serve as a biomarker of accumulated oxidative stress over the lifespan. Many studies have focused on understanding the contribution of DiY to AD pathogenesis and have revealed that DiY crosslinks can be found in both Aβ and tau deposits - the two key proteins involved in the formation of amyloid plaques and tau tangles, respectively. However, there is no consensus yet in the field on the impact of DiY on Aβ and tau function, aggregation, and toxicity. Here we review the current understanding of the role of DiY on Aβ and tau gathered over the last 20 years since the first observation, and discuss the effect of this modification for Aβ and tau aggregation, and its potential as a biomarker for AD.

Mechanical Properties of Protein-Based Hydrogels Derived from Binary Protein Mixtures-A Feasibility Study.

Hydrogels based on natural polymers such as proteins are considered biocompatible and, therefore, represent an interesting class of materials for application in the field of biomedicine and high-performance materials. However, there is a lack of understanding of the proteins which are able to form hydrogel networks by photoinduced dityrosine crosslinking as well as a profound knowledge of the formed network itself and the mechanisms which are responsible for the resulting mechanical properties of such protein-based hydrogels. In this study, casein, bovine serum albumin, α-amylase, and a hydrophobic elastin-like protein were used to prepare binary protein mixtures with defined concentration ratios. After polymerization, the mechanical properties of the resulting homopolymeric and copolymeric hydrogels were determined using rheological methods depending on the protein shares used. In additional uniaxial compression tests, the fracture strain was shown to be independent of the protein shares, while hydrogel toughness and compressive strength were increased for protein-based hydrogels containing casein.

Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) invitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.

Peptide Cross-Linking Using Tyrosine Residues Facilitated by an Exogenous Nickel-Histidine Complex: A Facile Approach for Enhancing Vaccine-Specific Immunogenicity.

An improved method for the generation of peptide vaccines using di-tyrosine cross-linking is described. The conserved ion channel peptide, M2e, of influenza A virus was modified with the addition of small tyrosine-rich regions (GYGY-) at both the N- and C-termini and extensively cross-linked via tyrosine-tyrosine linkages to form peptide nanoclusters. The cross-linking was catalyzed using exogenous nickel(II) ions complexed to an exogenous glycine-glycine-histidine peptide in the presence of an oxidizer. Mice that were intranasally or intramuscularly immunized with the M2e-vaccine nanoclusters induced comparable levels of M2e-specific serum antibodies. Vaccination via the intranasal or intramuscular route protected mice from subsequent lethal challenge with an influenza A virus. In comparison to our previous approach, where a histidine-rich tag was added into the peptide structure, the use of exogenous histidine reduced irrelevant off-target immune response. Additionally, the purity of the resulting nanoclusters is an attractive feature, making this approach appealing for vaccine development.

Bio-based material formulation for extrusion printing by dityrosine crosslinking of unmodified casein

In the development of new functional bio-based materials in the field of three-dimensional (3D) printing, visible light-induced dityrosine crosslinking gains increasing interest. In this context, most current bio-based materials and ink formulations rely on previously modified chemical substances with increased tyrosine availability. In contrast, we developed and characterized a photopolymerizable ink formulation for extrusion printing based on the unmodified and naturally occurring protein casein. Manufacturability of formulations containing protein, photoinitiating system, buffer and a thickening agent turned out to be a key factor for the ink development. In total, eight different thickening agents were assessed regarding their suitability to increase the viscosity of the ink formulation to expand the fabrication window for extrusion-based 3D printing. The mechanical properties of the ink formulation and hydrogel in presence of sodium alginate were further characterized and the macroscopic fabrication of auxetic structures consisting of up to 30 layers was achieved by applying extrusion-based printing.

Dityrosine Cross-links are Present in Alzheimer’s Disease-derived Tau Oligomers and Paired Helical Filaments (PHF) which Promotes the Stability of the PHF-core Tau (297–391) In Vitro

A characteristic hallmark of Alzheimer’s Disease (AD) is the pathological aggregation and deposition of tau into paired helical filaments (PHF) in neurofibrillary tangles (NFTs). Oxidative stress is an early event during AD pathogenesis and is associated with tau-mediated AD pathology. Oxidative environments can result in the formation of covalent dityrosine crosslinks that can increase protein stability and insolubility. Dityrosine cross-linking has been shown in Aβ plaques in AD and α-synuclein aggregates in Lewy bodies in ex vivo tissue sections, and this modification may increase the insolubility of these aggregates and their resistance to degradation. Using the PHF-core tau fragment (residues 297 – 391) as a model, we have previously demonstrated that dityrosine formation traps tau assemblies to reduce further elongation. However, it is unknown whether dityrosine crosslinks are found in tau deposits in vivo in AD and its relevance to disease mechanism is unclear. Here, using transmission electron microscope (TEM) double immunogold-labelling, we reveal that neurofibrillary NFTs in AD are heavily decorated with dityrosine crosslinks alongside tau. Single immunogold-labelling TEM and fluorescence spectroscopy revealed the presence of dityrosine on AD brain-derived tau oligomers and fibrils. Using the tau (297–391) PHF-core fragment as a model, we further showed that prefibrillar tau species are more amenable to dityrosine crosslinking than tau fibrils. Dityrosine formation results in heat and SDS stability of oxidised prefibrillar and fibrillar tau assemblies. This finding has implications for understanding the mechanism governing the insolubility and toxicity of tau assemblies in vivo.

Surface Biofunctionalization of Silk Biomaterials Using Dityrosine Cross-Linking.

Biofunctionalization of silk biomaterial surfaces with extracellular matrix (ECM) molecules, cell binding peptides, or growth factors is important in a range of applications, including tissue engineering and development of implantable medical devices. Passive adsorption is the most common way to immobilize molecules of interest on preformed silk biomaterials but can lead to random molecular orientations and displacement from the surface, limiting their applications. Herein, we developed techniques for covalent immobilization of biomolecules using enzyme- or photoinitiated formation of dityrosine bonds between the molecule of interest and silk. Using recombinantly expressed domain V of the human basement membrane proteoglycan perlecan (rDV) as a model molecule, we demonstrated that rDV can be covalently immobilized via dityrosine cross-linking without the need to modify rDV or silk biomaterials. Dityrosine-based immobilization resulted in a different molecular orientation to passively absorbed rDV with less C- and N-terminal region exposure on the surface. Dityrosine-based immobilization supported functional rDV immobilization where immobilized rDV supported endothelial cell adhesion, spreading, migration, and proliferation. These results demonstrate the utility of dityrosine-based cross-linking in covalent immobilization of tyrosine-containing molecules on silk biomaterials in the absence of chemical modification, adding a simple and accessible technique to the silk biofunctionalization toolbox.

Recombinant Rice Quiescin Sulfhydryl Oxidase Strengthens the Gluten Structure through Thiol/Disulfide Exchange and Hydrogen Peroxide Oxidation.

Recombinant rice quiescin sulfhydryl oxidase (rQSOX) has the potential to improve the flour processing quality, but the mechanisms remain unclear. The effects of rQSOX on bread quality, dough rheology, and gluten structure and composition, with glucose oxidase as a positive control, were investigated. rQSOX addition could improve the dough processing quality, as proved by enhanced viscoelastic properties of dough as well as a softer crumb, higher specific volume, and lower moisture loss of bread. These beneficial effects were attributed to gluten protein polymerization and gluten network strengthening, evidenced by the improved concentration of SDS-insoluble gluten and formation of large gluten aggregates and the increased α-helix and β-turn conformation. Furthermore, decreased free sulfhydryl and increased dityrosine in gluten as well as improved H2O2 content in dough suggested that the rQSOX dough strengthening mechanism was mainly based on the formation of disulfide bonds and dityrosine cross-links in gluten by both thiol/disulfide direct exchange and hydrogen peroxide indirect oxidation pathways.

Knockdown of E1- and E2-ubiquitin enzymes triggers defective chorion biogenesis and modulation of autophagy-related genes in the follicle cells of the vector Rhodnius prolixus.

In insects, the last stage of oogenesis is the process where the chorion layers (eggshell) are synthesized and deposited on the surface of the oocytes by the follicle cells. Protein homeostasis is determined by the fine-tuning of translation and degradation pathways, and the ubiquitin-proteasome system is one of the major degradative routes in eukaryotic cells. The conjugation of ubiquitin to targeted substrates is mediated by the ordered action of E1-activating, E2-conjugating, and E3-ligase enzymes, which covalently link ubiquitin to degradation-targeted proteins delivering them to the proteolytic complex proteasome. Here, we found that the mRNAs encoding polyubiquitin (pUbq), E1, and E2 enzymes are highly expressed in the ovaries of the insect vector of Chagas Disease Rhodnius prolixus. RNAi silencing of pUbq was lethal whereas the silencing of E1 and E2 enzymes resulted in drastic decreases in oviposition and embryo viability. Eggs produced by the E1- and E2-silenced insects presented particular phenotypes of altered chorion ultrastructure observed by high-resolution scanning electron microscopy as well as readings for dityrosine cross-linking and X-ray elemental microanalysis, suggesting a disruption in the secretory routes responsible for the chorion biogenesis. In addition, the ovaries from silenced insects presented altered levels of autophagy-related genes as well as a tendency of upregulation in ER chaperones, indicating a disturbance in the general biosynthetic-secretory pathway. Altogether, we found that E1 and E2 enzymes are essential for chorion biogenesis and that their silencing triggers the modulation of autophagy genes suggesting a coordinated function of both pathways for the progression of choriogenesis.

Photo-enzymatic dityrosine crosslinking for bioprinting

In this research work, we demonstrate photo-triggering of the horseradish peroxidase (HRP) dityrosine crosslinking. The photo-enzymatic crosslinking of tyramine modified alginate was obtained by introducing riboflavin as a photosensitizer, and by exposing the polymer solution to light radiation at 365 nm. The activation of HRP by light was confirmed by photo-rheology. This crosslinking method proved to be superior to UV crosslinking by riboflavin alone both in terms of kinetics and obtainable storage modulus. Furthermore, activation of HRP by UV light offers the possibility to trigger the onset of the reaction in a precise manner, which is not available with the traditional use of hydrogen peroxide as the activator. The photo-enzymatic crosslinking was applied to bioprinting, to verify its potential in biofabrication techniques requiring precise on-demand crosslinking of hydrogels.