Synthetic biology in microalgae towards fucoxanthin production for pharmacy and nutraceuticals.

Synthetic biology has emerged as a powerful tool for engineering biological systems to produce valuable compounds, including pharmaceuticals and nutraceuticals. Microalgae, in particular, offer a promising platform for the production of bioactive compounds due to their high productivity, low land and water requirements, and ability to perform photosynthesis. Fucoxanthin, a carotenoid pigment found predominantly in brown seaweeds and certain microalgae, has gained significant attention in recent years due to its numerous health benefits, such as antioxidation, antitumor effect and precaution osteoporosis. This review provides an overview of the principles and applications of synthetic biology in the microbial engineering of microalgae for enhanced fucoxanthin production. Firstly, the fucoxanthin bioavailability and metabolism in vivo was introduced for the beneficial roles, followed by the biological functions of anti-oxidant activity, anti-inflammatory activity, antiapoptotic role antidiabetic and antilipemic effects. Secondly, the cultivation condition and strategy were summarized for fucoxanthin improvement with low production costs. Thirdly, the genetic engineering of microalgae, including gene overexpression, knockdown and knockout strategies were discussed for further improving the fucoxanthin production. Then, synthetic biology tools of CRISPR-Cas9 genome editing, transcription activator-like effector nucleases as well as modular assembly and chassis engineering were proposed to precise modification of microalgal genomes to improve fucoxanthin production. Finally, challenges and future perspectives were discussed to realize the industrial production and development of functional foods of fucoxanthin from microalgae.

Characterization of an ablative fractional CO2 laser-induced wound-healing model based on in vitro 3D reconstructed skin.

OBJECTIVE: This study describes the development and characterization of a novel in vitro wound-healing model based on a full-thickness reconstructed skin by exposing the tissue to fractional ablative laser treatment. METHOD: A 3D full-thickness skin model was fabricated and treated with fractional ablative CO2 laser. Wound-healing process was characterized by HE staining, noninvasive OCT imaging, immunostaining, as well as transepidermal water loss measurement. Cytokines and proteins involved in the inflammatory and dermal remodeling process were studied by ELISA and protein array assays. RESULTS: Fractional ablative CO2 treatment induced a wound zone of 9 mm in diameter, containing 56 micro-wounds with 200 µm diameter and 500-700 µm in depth on reconstructed full-thickness skin model. HE staining revealed a typical wound morphology and healing process with migration of keratinocytes, formation and extrusion of necrotic tissue, and cell inclusion in dermis, which correlates with clinical observations. Based on OCT and TEWL measurements, the re-epithelialization took place over 2 days. Laser-triggered keratinocytes proliferation and differentiation were demonstrated by activated Ki67 and Filaggrin expression respectively. Injury-invoked cytokine ICAM-1 showed instant upregulation on Day 1. Decreased epidermis thickness and depression of IGFBP-2 protein level synergistically indicated the unavoidable thermal side effects from laser treatment. Downregulated DKK-1 protein level and upregulation of α-SMA together implicated the risk of potential fibrosis post-laser treatment. CONCLUSION: This in vitro laser wounded reconstructed skin model captured the key events of wound-healing process, could be used to investigate the mechanisms of wound-healing triggered by a commonly used beauty procedure, and also provides a valuable tool for evaluating the efficacy of novel actives for the post-procedure application.

COVID-19-induced layoff, survivors' COVID-19-related stress and performance in hospitality industry: The moderating role of social support.

The COVID-19 pandemic has hit the hospitality industry hard globally, resulting in millions of employees being laid off. Drawing upon the conservation of resources theory, this study aims to empirically examine how and when COVID-19-induced layoff influences employees' in-role and extra-role performance in the hospitality industry. We tested this model by using field data collected from 302 employees and their supervisors in China across two waves. Results revealed that COVID-19-induced layoff increases survivors' COVID-19-related stress, which in turn leads to decreased in-role and extra-role performance. The strength of these indirect effects is mitigated by perceived family support against COVID-19. Unexpectedly, perceived organizational support against COVID-19 intensifies these indirect effects. The theoretical and practical implications of this study are further discussed.

Ocular phenotype of Fbn2-null mice.

PURPOSE: Fibrillin-2 (Fbn2) is the dominant fibrillin isoform expressed during development of the mouse eye. To test its role in morphogenesis, we examined the ocular phenotype of Fbn2(-/-) mice. METHODS: Ocular morphology was assessed by confocal microscopy using antibodies against microfibril components. RESULTS: Fbn2(-/-) mice had a high incidence of anterior segment dysgenesis. The iris was the most commonly affected tissue. Complete iridal coloboma was present in 37% of eyes. Dyscoria, corectopia and pseudopolycoria were also common (43% combined incidence). In wild-type (WT) mice, fibrillin-2-rich microfibrils are prominent in the pupillary membrane (PM) during development. In Fbn2-null mice, the absence of Fbn2 was partially compensated for by increased expression of fibrillin-1, although the resulting PM microfibrils were disorganized, compared with WTs. In colobomatous adult Fbn2(-/-) eyes, the PM failed to regress normally, especially beneath the notched region of the iris. Segments of the ciliary body were hypoplastic, and zonular fibers, although relatively plentiful, were unevenly distributed around the lens equator. In regions where the zonular fibers were particularly disturbed, the synchronous differentiation of the underlying lens fiber cells was affected. CONCLUSIONS: Fbn2 has an indispensable role in ocular morphogenesis in mice. The high incidence of iris coloboma in Fbn2-null animals implies a previously unsuspected role in optic fissure closure. The observation that fiber cell differentiation was disturbed in Fbn2(-/-) mice raises the possibility that the attachment of zonular fibers to the lens surface may help specify the equatorial margin of the lens epithelium.

Development, composition, and structural arrangements of the ciliary zonule of the mouse.

PURPOSE: Here, we examined the development, composition, and structural organization of the ciliary zonule of the mouse. Fibrillin 1, a large glycoprotein enriched in force-bearing tissues, is a prominent constituent of the mouse zonule. In humans, mutations in the gene for fibrillin 1 (FBN1) underlie Marfan syndrome (MS), a disorder characterized by lens dislocation and other ocular symptoms. METHODS: Fibrillin expression was analyzed by in situ hybridization. The organization of the zonule was visualized using antibodies to Fbn1, Fbn2, and microfibril-associated glycoprotein-1 (Magp1) in conjunction with 5-ethynyl-2'-deoxyuridine (EdU), an S-phase marker. RESULTS: Microfibrils, enriched in Fbn2 and Magp1, were prominent components of the temporary vascular tunic of the embryonic lens. Fbn2 expression by nonpigmented ciliary epithelial cells diminished postnatally and there was a concomitant increase in Fbn1 expression, especially in cells located in valleys between the ciliary folds. Zonular fibers projected from the posterior pars plicata to the lens in anterior, equatorial, and posterior groupings. The attachment point of the posterior zonular fibers consisted of a dense meshwork of radially oriented microfibrils that we termed the fibrillar girdle. The fibrillar girdle was located directly above the transition zone, a region of the lens epithelium in which cells commit to terminal differentiation. CONCLUSIONS: The development and arrangement of the murine ciliary zonule are similar to those of humans, and consequently the mouse eye may be a useful model in which to study ocular complications of MS.

Shape of tropoelastin, the highly extensible protein that controls human tissue elasticity.

Elastin enables the reversible deformation of elastic tissues and can withstand decades of repetitive forces. Tropoelastin is the soluble precursor to elastin, the main elastic protein found in mammals. Little is known of the shape and mechanism of assembly of tropoelastin as its unique composition and propensity to self-associate has hampered structural studies. In this study, we solve the nanostructure of full-length and corresponding overlapping fragments of tropoelastin using small angle X-ray and neutron scattering, allowing us to identify discrete regions of the molecule. Tropoelastin is an asymmetric coil, with a protruding foot that encompasses the C-terminal cell interaction motif. We show that individual tropoelastin molecules are highly extensible yet elastic without hysteresis to perform as highly efficient molecular nanosprings. Our findings shed light on how biology uses this single protein to build durable elastic structures that allow for cell attachment to an appended foot. We present a unique model for head-to-tail assembly which allows for the propagation of the molecule's asymmetric coil through a stacked spring design.

Synthetic elastin hydrogels that are coblended with heparin display substantial swelling, increased porosity, and improved cell penetration.

Synthetic elastin hydrogels are useful tissue engineering scaffolds because they present cell binding sequences and display physical performance similar to that of human elastic tissue. Small pores and a low porosity can limit cellular penetration into elastin scaffolds. To overcome this problem, glycosaminoglycans were coblended with tropoelastin during the formation of synthetic elastin hydrogels. Heparin and dermatan sulfate increased the pore size and porosity of the hydrogels. Heparin was particularly effective as it enlarged the pore size from 6.6 ± 2.1 µm to 23.8 ± 8.5 µm, and generated structures occasionally separated by finely fenestrated thin walls, which allowed human dermal fibroblast cells to migrate as deep as ∼300 µm into the hydrogel under diffusion-limiting static culture conditions. Most cells displayed spindle-like morphology, appeared histologically normal and presented intact nuclei, as expected for a viable population. Hydrogel swelling studies showed that each of the hydrogels contracted as the temperature was raised from 4°C to 37°C; synthetic elastin-heparin was least affected by temperature with a contraction of only 22.4 ± 1.2%, which would facilitate its transition from cold storage to body temperature. All hydrogels displayed similar compression moduli of 5.5 ± 0.4 to 6.9 ± 0.6 kPa. Compressive elastic energy losses for synthetic elastin-heparin and synthetic elastin were 33.7 ± 1.3% and 31.7 ± 2.2% respectively.

Stages in tropoelastin coalescence during synthetic elastin hydrogel formation.

Synthetic human tropoelastin was chemically cross-linked to form elastic hydrogel-like structures in vitro. Discrete stages were identified during elastic hydrogel formation by cross-linking tropoelastin with bis(sulfosuccinimidyl) suberate at a range of protein concentrations during this process. In the early stages of this process, particles with the same dimensions as tropoelastin were seen. As hydrogel formation progressed, monomer width fibres were also observed. Overall, four distinct stages were identified: (1) tropoelastin monomers form discrete particles in the order of 200 nm diameter, (2) these particles merge to form larger spheres, (3) spheres coalesce into open linked networks, (4) coalesced spheres consolidate to form a porous structure to give synthetic elastin hydrogels.

Transient tropoelastin nanoparticles are early-stage intermediates in the coacervation of human tropoelastin whose aggregation is facilitated by heparan sulfate and heparin decasaccharides.

Tropoelastin assembly is a key step in the formation of elastin. We consider how nanoscale intracellular assemblies of tropoelastin can congregate in an extracellular environment to give microscale aggregates. We describe novel 200-300 nm spherical particles that serve as intermediates in the formation of the coacervate. Their aggregation gives 800 nm to 1 microm species. This process is facilitated by heparan sulfate and dermatan sulfate interactions which effectively lower the critical concentration to facilitate this transition. This coacervation process was examined using a panel of heparin chains of various lengths and showed greatest efficacy for the decasaccharide, followed by the octasaccharide, while the hexasaccharide displayed the shortest efficacious length. We propose that these oligosaccharide interactions enable the charge-mediated aggregation of positively charged tropoelastin. This biochemistry models glycosaminoglycan interactions on the cell surface during elastogenesis which is characterized by the clustering of nascent tropoelastin aggregates to form micron-sized spherules.

"Setting paint" analogy for the hydrophobic self-association of tropoelastin into elastin-like hydrogel.

Alkaline tropoelastin solutions (pH 11) were optically clear at low temperatures, but a firm gel formed when the temperature was raised to 37 degrees C. Reversion to a clear solution took place if the temperature was lowered to below 20 degrees C within less than 2 h, but not if 37 degrees C was maintained for several hours. The precipitated elastin-like hydrogel thus formed did not visually redissolve at low temperatures. Tropoelastin hydrogel was stable to subsequent washings with alkaline solution at 37 degrees C, but at 4 degrees C some hydrogel redissolved showing that association is at least partly reversible. Washing the hydrogel with neutral 8M urea solution at 4 degrees C dissolved less than 10% of tropoelastin in 24 h. We characterized this phenomenon by combining temperature-controlled light microscopy analysis, 1H NMR spectroscopy (temperature, diffusion, and relaxation time studies), and UV-absorption-based concentration measurements. The self-association of tropoelastin at pH 11 is due to hydrophobic interactions in an emulsion-like system in which the spherules coalesce in a manner like a water-based latex paint that forms a durable hydrophobic sheet as water and the organic solvent evaporate. In the present case, the sedimentation and entanglement of the tropoelastin porous sheets means that reverse dissolution is a kinetically slow process.

In situ polymerization of tropoelastin in the absence of chemical cross-linking.

Tropoelastin, the polypeptide monomer precursor of elastin, is covalently cross-linked to give stable elastic structures. We show here that elastic biomaterials can be generated from tropoelastin in the absence of the classically accepted cross-linking pathway. Under alkaline conditions tropoelastin proceeds through a sol-gel transition leading to the formation of an irreversible hydrogel. This does not occur at neutral pH. The resulting biomaterial is stable, elastic and flexible. Scanning electron microscopy revealed that the hydrogel forms through the coalescence of approximately 1 microm quantized protein spheres. These spheres resemble the tropoelastin-rich globules that accumulate on cultured cell surfaces during elastin formation. In vitro cell culture studies demonstrate that the hydrogel can support human skin fibroblast proliferation. In vivo studies demonstrate that following injection, the tropoelastin solution undergoes rapid localized gelation to form a persistent mass. These subcutaneous rodent injection data establish the material's potential as a novel cell-compatible elastic scaffold that can be formed in situ.

Glycosaminoglycan-mediated coacervation of tropoelastin abolishes the critical concentration, accelerates coacervate formation, and facilitates spherule fusion: implications for tropoelastin microassembly.

Elastogenesis and elastin repair depend on the secretion of tropoelastin from the cell, yet cellular production is low in the many biological systems that have been studied. To address the apparent paradox of a paucity of tropoelastin for cell surface microassembly, we examined the effects of the glycosaminoglycans heparin, heparan sulfate, and chondroitin sulfate B, on tropoelastin aggregate formation through coacervation. We found a significant effect, particularly of heparin, on the minimum or critical concentration of tropoelastin, which was required for microassembly, lowering critical concentration to a point that it was no longer detectable. The assemblies resulted in protein droplet formation that was visually indistinguishable from the spherules that typify coacervation. The spherules readily coalesced in the presence of heparin and higher concentrations of tropoelastin, resulting in an almost continuous layer of coacervated tropoelastin. Four stages of droplet behavior were observed: early droplet formation, approximately 6 mum droplet formation, and fusion of droplets followed by the formation of a coalesced layer. We conclude that glycosaminoglycans in the extracellular matrix have the capacity to promote coacervation at low concentrations of tropoelastin.