Macular pigment-enriched oil production from genome-edited microalgae.

BACKGROUND: The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain. RESULTS: Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93 ± 0.22 mg g-1 DCW: dried cell weight), zeaxanthin (3.12 ± 0.30 mg g-1 DCW), and lipids (450.09 ± 25.48 mg g-1 DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196 ± 20.1 mg g-1 DCW of edible microalgal oil containing 8.42 ± 0.92 mg g-1 lutein of oil and 7.69 ± 1.03 mg g-1 zeaxanthin of oil was produced. CONCLUSION: Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.

CRISPR/Cas9-Mediated Knockout of the Lycopene ε-Cyclase for Efficient Astaxanthin Production in the Green Microalga Chlamydomonas reinhardtii.

Carotenoids are valuable pigments naturally occurring in all photosynthetic plants and microalgae as well as in selected fungi, bacteria, and archaea. Green microalgae developed a complex carotenoid profile suitable for efficient light harvesting and light protection and harbor great capacity for carotenoid production through the substantial power of the endogenous 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Previous works established successful genome editing and induced significant changes in the cellular carotenoid content in Chlamydomonas reinhardtii. This study employs a tailored carotenoid pathway for engineered bioproduction of the valuable ketocarotenoid astaxanthin. Functional knockout of lycopene ε-cyclase (LCYE) and non-homologous end joining (NHEJ)-based integration of donor DNA at the target site inhibit the accumulation of α-carotene and consequently lutein and loroxanthin, abundant carotenoids in C. reinhardtii without changes in cellular fitness. PCR-based screening indicated that 4 of 96 regenerated candidate lines carried (partial) integrations of donor DNA and increased ß-carotene as well as derived carotenoid contents. Iterative overexpression of CrBKT, PacrtB, and CrCHYB resulted in a 2.3-fold increase in astaxanthin accumulation in mutant ΔLCYE#3 (1.8 mg/L) compared to the parental strain UVM4, which demonstrates the potential of genome editing for the design of a green cell factory for astaxanthin bioproduction.

Photoautotrophic cultivation of a Chlamydomonas reinhardtii mutant with zeaxanthin as the sole xanthophyll.

BACKGROUND: Photosynthetic microalgae are known for their sustainable and eco-friendly potential to convert carbon dioxide into valuable products. Nevertheless, the challenge of self-shading due to high cell density has been identified as a drawback, hampering productivity in sustainable photoautotrophic mass cultivation. To address this issue, mutants with altered pigment composition have been proposed to allow a more efficient light diffusion but further study on the role of the different pigments is still needed to correctly engineer this process. RESULTS: We here investigated the Chlamydomonas reinhardtii Δzl mutant with zeaxanthin as the sole xanthophyll. The Δzl mutant displayed altered pigment composition, characterized by lower chlorophyll content, higher chlorophyll a/b ratio, and lower chlorophyll/carotenoid ratio compared to the wild type (Wt). The Δzl mutant also exhibited a significant decrease in the light-harvesting complex II/Photosystem II ratio (LHCII/PSII) and the absence of trimeric LHCIIs. This significantly affects the organization and stability of PSII supercomplexes. Consequently, the estimated functional antenna size of PSII in the Δzl mutant was approximately 60% smaller compared to that of Wt, and reduced PSII activity was evident in this mutant. Notably, the Δzl mutant showed impaired non-photochemical quenching. However, the Δzl mutant compensated by exhibiting enhanced cyclic electron flow compared to Wt, seemingly offsetting the impaired PSII functionality. Consequently, the Δzl mutant achieved significantly higher cell densities than Wt under high-light conditions. CONCLUSIONS: Our findings highlight significant changes in pigment content and pigment-protein complexes in the Δzl mutant compared to Wt, resulting in an advantage for high-density photoautotrophic cultivation. This advantage is attributed to the decreased chlorophyll content of the Δzl mutant, allowing better light penetration. In addition, the accumulated zeaxanthin in the mutant could serve as an antioxidant, offering protection against reactive oxygen species generated by chlorophylls.

Nanoparticle Encapsulation of the Hexane Fraction of Cyperus Rotundus Extract for Enhanced Antioxidant and Anti-Inflammatory Activities in vitro.

Aim: Cyperus rotundus L. (CR) is traditionally used in medicine for its anti-inflammatory properties. In particular, α-cyperone, which is isolated from the essential oil and found primarily in the n-hexane fraction of the ethanolic extract, is known to inhibit NO production in LPS-stimulated RAW 264.7 cells. However, high concentrations of α-cyperone are required for sufficient anti-inflammatory activity. Even, essential oil obtained from C. rotundus has the disadvantage of low solubility and stability in aqueous environment, which makes it difficult to be applied in various fields and easily loses its activity. Therefore, in this study, we aimed to increase the extraction yield of C. rotundus by microbubble extraction and prepare nanoparticles (NPs) that can preserve its activity in a stable and bioavailable manner by utilizing nanoprecipitation. Methods: C. rotundus rhizomes were extracted in 50% ethanol using microbubbles and then fractionated with n-hexane to obtain α-cyperone-rich C. rotundus n-hexane fraction (CRHF). The biodegradable plant extract, α-cyperone, was prepared as green nanoparticles (CR@NPs) by nanoprecipitation technique under mild reaction conditions. The physicochemical properties of CR@NPs, including size, polydispersity index, and surface charge, were determined using dynamic light scattering. The extraction yield and encapsulation efficiency of α-cyperone were quantified by high-performance liquid chromatography. Antioxidant and anti-inflammatory activities were evaluated by DPPH assay and in vitro ROS and NO assays, and biocompatibility was assessed by MTT assay. Results: C. rotundus loaded nanoparticles demonstrated overcoming the limitation of α-cyperone solubility and stability in CRHF and also the antioxidant, anti-inflammatory properties as evidenced by in vitro assays in cellular models. Conclusion: The versatility of green chemistry, such as α-cyperone, enables the production of nanoparticles with promising biomedical applications such as cosmetics, pharmaceuticals, and food products.

Genome engineering via gene editing technologies in microalgae.

CRISPR-Cas has revolutionized genetic modification with its comparative simplicity and accuracy, and it can be used even at the genomic level. Microalgae are excellent feedstocks for biofuels and nutraceuticals because they contain high levels of fatty acids, carotenoids, and other metabolites; however, genome engineering for microalgae is not yet as developed as for other model organisms. Microalgal engineering at the genetic and metabolic levels is relatively well established, and a few genomic resources are available. Their genomic information was used for a "safe harbor" site for stable transgene expression in microalgae. This review proposes further genome engineering schemes including the construction of sgRNA libraries, pan-genomic and epigenomic resources, and mini-genomes, which can together be developed into synthetic biology for carbon-based engineering in microalgae. Acetyl-CoA is at the center of carbon metabolic pathways and is further reviewed for the production of molecules including terpenoids in microalgae.

Effect of Plantago asiatica L. extract on the anagen phase in human hair follicle dermal papilla cells.

BACKGROUND: The hair growth cycle consists of the anagen, catagen, and telogen phases, and hair follicle dermal papilla (HDP) cells of human hair play a role in the initiation and maintenance of the anagen phase. Reduction in HDP cells contributes to hair loss; however, the limited treatment options are associated with negative side effects. Therefore, a naturally derived substance with hair loss-preventing properties is needed. AIM: We investigated the hair growth-stimulating activities of Plantago asiatica L. extract (PAE) and its molecular mechanism in HDP cells. METHODS: Cell proliferation was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution. Relative mRNA and protein expression levels of hair growth factors were determined using quantitative real-time polymerase chain reaction and western blotting, respectively. Additionally, a tube formation assay was performed in human umbilical vein endothelial cells (HUVEC). RESULTS: Plantago asiatica L. extract significantly increased the cell proliferation and expression of hair growth factors, including keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2) and MYC, in HDP cells. Moreover, PAE led to the accumulation of ß-catenin by promoting the phosphorylation of glycogen synthase kinase-3 beta (GSK-3ß) at Ser9 and cAMP response element-binding protein (CREB) at Ser133 via phosphorylation of extracellular signal-regulated kinase (ERK) (Thr202/Tyr204). PAE also increased tube formation in HUVECs, which promoted angiogenesis for the anagen phase. CONCLUSIONS: Plantago asiatica L. extract amplified tube formation and production of growth factors (KGF, VEGF) via the activation of GSK-3ß/ß-catenin and mitogen-activated protein kinase (MAPK)/CREB signaling pathways, demonstrating its potential to safely promote hair growth by inducing the anagen phase.

Flexible and Transparent Electrode Based on Ag-Nanowire Embedded Colorless Poly(amide-imide).

Graphene oxide-cysteamine-silver nanoparticle (GCA)/silver nanowire (AgNW)/GCA/colorless poly(amide-imide) (cPAI) structures based on cPAI substrates with polyimide and polyamide syntheses were fabricated to study their characteristics. A layer of electrodes was constructed using a sandwich structure-such as GCA/AgNW/GCA-with cPAI used as a substrate to increase the heat resistance and improve their mechanical properties. Furthermore, to overcome the disadvantages of AgNWs-such as their high surface roughness and weak adhesion between the substrate and electrode layers-electrodes with embedded structures were fabricated using a peel-off process. Through bending, tapping, and durability tests, it was confirmed that these multilayer electrodes exhibited better mechanical durability than conventional AgNW electrodes. Resistive random-access memory based on GCA/AgNW/GCA/cPAI electrodes was fabricated, and its applicability to nonvolatile memory was confirmed. The memory device had an ON/OFF current ratio of ~104@0.5 V, exhibiting write-once-read-many time characteristics, maintaining these memory characteristics for up to 300 sweep cycles. These findings suggest that GCA/AgNW/GCA/cPAI electrodes could be used as flexible and transparent electrodes for next-generation flexible nonvolatile memories.

Quantitative analysis of adsorption and desorption of volatile organic compounds on reusable zeolite filters using gas chromatography.

In this study, we propose a method to quantitatively analyze the concentration of VOCs adsorbed on zeolite filters via gas chromatography (GC). The sampled VOCs from the filters with ethanol as a solution were characterized using GC to determine the concentration of the adsorbed VOCs by comparing the areas of GC peaks of the detected VOCs and ethanol. The proposed method also enabled determination of the desorption (regeneration) conditions of the zeolite filters according to heating temperature and time for various VOCs. Repeated adsorption and desorption of VOCs on zeolite filters and GC analyses allow us to evaluate the durability and reusability of the filter and could help predict the lifetime of zeolite filters in practice.

Flexible Transparent Electrode Characteristics of Graphene Oxide/Cysteamine/AgNP/AgNW Structure.

Graphene oxide (GO)-cysteamine-Ag nanoparticles (GCA)-silver nanowire (AgNW) fabricated by depositing GCA over sprayed AgNWs on PET films were proposed for transparent and flexible electrodes, and their optical, electrical, and mechanical properties were analyzed by energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning electron microscopy, transmission electron microscopy, current-voltage measurements, and bending test. GCA-AgNW electrodes show optical transmittance of >80% at 550 nm and exhibit a high figure-of-merit value of up to 116.13 in the samples with sheet resistances of 20-40 Ω/◻. It was observed that the detrimental oxidation of bare AgNWs over time was considerably decreased, and the mechanical robustness was improved. To apply the layer as an actual electrode in working devices, a Pt/GO/poly(3,4-ethylenedioxythiophene) polystyrene sulfonate/GCA-AgNW/polyethylene terephthalate structure was fabricated, and resistive switching memory was demonstrated. On the basis of these results, we confirm that the proposed GCA-AgNW layer can be used as transparent and flexible electrode.

Ni-Cu Nanoparticles-Embedded Ag-Based Reflector for High-Efficiency Light-Emitting Diodes.

We investigated the use of a silver reflector embedded with Ni-Cu nanoparticles to achieve low resistance and high reflectivity in GaN-based flip-chip light-emitting diodes. Compared to a single layer of Ag, the NC-NPs/Ag reflector exhibits a higher light reflectance of ~90% at a wavelength of 450 nm, a lower contact resistance of 4.75 × 10-5 II cm², and improved thermal stability after annealing at 400°C. The NC-NPs formed after the annealing process prevents agglomeration of the Ag layer, while also reducing the Schottky barrier height between the p-GaN layer and metal reflector. The LED fabricated with a NC-NPs/Ag reflector exhibited a forward-bias voltage of 3.13 V and an improvement in light output power of 36.6% (at 20 mA), when compared with the LED composed of a Ag SL. This result indicates that the NC-NPs/Ag reflector is a promising p-type reflector for high-intensity light-emitting diodes.

Note: Temperature compensation of time-of-flight light detection and ranging (LiDAR) using feedback control of signal-to-noise ratio of photodetector.

Time-of-flight-based two-dimensional and three-dimensional light detection and ranging (LiDAR) applications have recently been implemented in several industries because of their high-precision measuring capabilities over long distances in outdoor environments. Avalanche photodetectors (APDs) are commonly used for LiDARs because of their high internal gain that significantly amplifies a measured signal. However, the magnitude of the measured signal changes significantly with temperature variations, owing to the temperature dependent characteristics of the APD. In this study, a temperature compensation method, in which a bias voltage of the APD is adjusted for temperature changes using signal-to-noise ratio feedback control, is proposed to solve the problem. This method has the advantage of a simple hardware configuration, without using a conventionally considered cooler.

Preparation of Polyimide/Graphene Oxide Nanocomposite and Its Application to Nonvolatile Resistive Memory Device.

2,6-Diaminoanthracene (AnDA)-functionalized graphene oxide (GO) (AnDA-GO) was prepared and used to synthesize a graphene oxide-based polyimide (PI-GO) by the in-situ polymerization method. A PI-GO nanocomposite thin film was prepared and characterized by infrared (IR) spectroscopy, thermogravimetric analysis (TGA) and UV-visible spectroscopy. The PI-GO film was used as a memory layer in the fabrication of a resistive random access memory (RRAM) device with aluminum (Al) top and indium tin oxide (ITO) bottom electrodes. The device showed write-once-read-many-times (WORM) characteristics with a high ON/OFF current ratio (Ion/Ioff = 3.41 × 108). This excellent current ratio was attributed to the high charge trapping ability of GO. In addition, the device had good endurance until the 100th cycle. These results suggest that PI-GO is an attractive candidate for applications in next generation nonvolatile memory.

Note: Optical and electronic design of an amplitude-modulated continuous-wave laser scanner for high-accuracy distance measurement.

To utilize a time-of-flight-based laser scanner as a distance measurement sensor, the measurable distance and accuracy are the most important performance parameters to consider. For these purposes, the optical system and electronic signal processing of the laser scanner should be optimally designed in order to reduce a distance error caused by the optical crosstalk and wide dynamic range input. Optical system design for removing optical crosstalk problem is proposed in this work. Intensity control is also considered to solve the problem of a phase-shift variation in the signal processing circuit caused by object reflectivity. The experimental results for optical system and signal processing design are performed using 3D measurements.

Note: continuous-wave time-of-flight laser scanner using two laser diodes to avoid 2π ambiguity.

In the continuous-wave time-of-flight method, the distance traveled by light can be obtained by using the phase difference between the reference signal and the measured signal. However, when the phase difference exceeds 2π, the distance cannot be differentiated because the distance variation repeats every 2π period. In this paper, we propose a method in which low- and high-frequency signals are separately applied to two laser diodes simultaneously, and processed using two different signal processing circuits to solve the 2π ambiguity problem and achieve a high distance resolution for a longer distance measurement.

Note: Intensity control of a phase-shift based laser scanner for reducing distance errors caused by varying surface reflectivity.

In the phase-shift measurement method, the distance traveled by light can be obtained on the basis of the phase difference between the reference signal and the measured signal. When a different colored object is measured, the intensity of the measured signal varies greatly, even at the same distance, which causes a different phase delay owing to the wide dynamic range input into the signal processing circuit. In this study, an intensity control method is proposed to solve this phase delay problem.