Genome editing in macroalgae: advances and challenges.

This minireview examines the current state and challenges of genome editing in macroalgae. Despite the ecological and economic significance of this group of organisms, genome editing has seen limited applications. While CRISPR functionality has been established in two brown (Ectocarpus species 7 and Saccharina japonica) and one green seaweed (Ulva prolifera), these studies are limited to proof-of-concept demonstrations. All studies also (co)-targeted ADENINE PHOSPHORIBOSYL TRANSFERASE to enrich for mutants, due to the relatively low editing efficiencies. To advance the field, there should be a focus on advancing auxiliary technologies, particularly stable transformation, so that novel editing reagents can be screened for their efficiency. More work is also needed on understanding DNA repair in these organisms, as this is tightly linked with the editing outcomes. Developing efficient genome editing tools for macroalgae will unlock the ability to characterize their genes, which is largely uncharted terrain. Moreover, given their economic importance, genome editing will also impact breeding campaigns to develop strains that have better yields, produce more commercially valuable compounds, and show improved resilience to the impacts of global change.

Interactive Effects of Blue Light and Water Turbulence on the Growth of the Green Macroalga Ulva australis (Chlorophyta).

Macroalgal growth and yield are key to sustainable aquaculture. Although light and water turbulence are two important factors that affect algal productivity, research on their interaction is limited. Therefore, in this study, we investigated the effects of different wavelengths of light and the presence or absence of water turbulence on the growth of the green macroalga Ulva australis. Water turbulence was found to enhance the growth of U. australis irrespective of photosynthetic performance, but only in blue light cultures. The quantum dose of blue light required to induce 50% growth promotion was 1.02 mol m-2, which is comparable to the reported values for cryptochrome-mediated effects in other macroalgae. The combined effect of blue light and water turbulence led to the accumulation of photosynthesis-related proteins that support plastid differentiation and facilitate efficient photosynthesis and growth. Our findings thus highlight the potential of harnessing blue light and water turbulence to maximise macroalgal cultivation for sustainable and profitable algal aquaculture.

Extracellular Vimentin Alters Energy Metabolism And Induces Adipocyte Hypertrophy.

BACKGRUOUND: Previous studies have reported that oxidative stress contributes to obesity characterized by adipocyte hypertrophy. However, mechanism has not been studied extensively. In the current study, we evaluated role of extracellular vimentin secreted by oxidized low-density lipoprotein (oxLDL) in energy metabolism in adipocytes. METHODS: We treated 3T3-L1-derived adipocytes with oxLDL and measured vimentin which was secreted in the media. We evaluated changes in uptake of glucose and free fatty acid, expression of molecules functioning in energy metabolism, synthesis of adenosine triphosphate (ATP) and lactate, markers for endoplasmic reticulum (ER) stress and autophagy in adipocytes treated with recombinant vimentin. RESULTS: Adipocytes secreted vimentin in response to oxLDL. Microscopic evaluation revealed that vimentin treatment induced increase in adipocyte size and increase in sizes of intracellular lipid droplets with increased intracellular triglyceride. Adipocytes treated with vimentin showed increased uptake of glucose and free fatty acid with increased expression of plasma membrane glucose transporter type 1 (GLUT1), GLUT4, and CD36. Vimentin treatment increased transcription of GLUT1 and hypoxia-inducible factor 1α (Hif-1α) but decreased GLUT4 transcription. Adipose triglyceride lipase (ATGL), peroxisome proliferator-activated receptor γ (PPARγ), sterol regulatory element-binding protein 1 (SREBP1), diacylglycerol O-acyltransferase 1 (DGAT1) and 2 were decreased by vimentin treatment. Markers for ER stress were increased and autophagy was impaired in vimentin-treated adipocytes. No change was observed in synthesis of ATP and lactate in the adipocytes treated with vimentin. CONCLUSION: We concluded that extracellular vimentin regulates expression of molecules in energy metabolism and promotes adipocyte hypertrophy. Our results show that vimentin functions in the interplay between oxidative stress and metabolism, suggesting a mechanism by which adipocyte hypertrophy is induced in oxidative stress.

Assessing industrial wastewater effluent toxicity using boosting algorithms in machine learning: A case study on ecotoxicity prediction and control strategy development.

Trace heavy metals have a tendency to persist in the effluent of industrial wastewater treatment facilities, leading to toxic effects on downstream water bodies. Traditional assessment methods relied on animal testing, but ethical concerns have rendered them unacceptable. An alternative solution is to evaluate wastewater toxicity using trophic-level aquatic organisms as bioassays. However, these bioassay methods involve costly and time-consuming chemical and biological analytical experiments. In this study, an artificial intelligence-powered water quality assessment (AiWA) approach is proposed for predicting industrial effluent ecotoxicity to further enhance the quick and cost-effective ecotoxicity assessment process. Initially, 99 samples were collected from industrial wastewater treatment plants representing 21 different industries in the Republic of Korea. Fourteen parameters were measured, encompassing both physicochemical and ecotoxicological aspects. Boosting algorithms, especially extreme gradient boosting (XGBoost) and adaptive boosting (AdaBoost), were employed for model development. XGBoost outperformed AdaBoost in terms of model performance. Feature selection analysis revealed that conductivity, copper, lead, selenium, pH, and zinc concentrations were the most suitable inputs for training the boosting model. The innovated XGBoost-based AiWA model demonstrated significantly higher performance (i.e., up to 80%) compared to conventional models with an R2 value of exceeding 0.94 and root mean square error of 3.5 toxicity unit for predicting the integrated toxicity unit (ITU). Additionally, pH and conductivity emerged as crucial indicators for reflecting ecotoxicity levels. Specially, this case study indicated that non-toxic/directly dischargeable levels (TU ≤ 1) were achieved when the pH ranged from 6.8 to 8.4 and the conductivity remained below 1651 µS/cm. These findings are expected to facilitate rapid and cost-effective detection of heavy metal ecotoxicity in industrial wastewater effluents, aiding decision-making in wastewater management.

Giant Duckweed (Spirodela polyrhiza) Root Growth as a Simple and Sensitive Indicator of Copper and Chromium Contamination.

Aquatic environment are often contaminated with heavy metals from various industrial sources. However, physicochemical techniques for pollutant detection are limited, thus prompting the need for additional bioassays. We investigated the use of greater duckweed (Spirodela polyrhiza) as a bioindicator of metal pollution. We exposed S. polyrhiza to four pollutants (namely, silver, cadmium, copper, and chromium) and assessed metal toxicity by measuring its frond area and the length of its regrown roots. The plant displayed significant differences in both frond size and root growth in response to the four metals. Silver was the most toxic (EC50 = 23 µg L-1) while copper the least (EC50 = 365-607 µg L-1). Direct comparisons of metal sensitivity and the reliability of the two endpoint assays showed that root growth was more sensitive (lower in terms of 50% effective concentration) to chromium, cadmium, and copper, and was more reliable (lower in terms of coefficient of variation) than those for frond area. Compared to conventional Lemna-based tests, the S. polyrhiza test is easier to perform (requiring only one 24-well plate, 3 mL of medium and a 72-h exposure). Moreover, it does not require livestock cultivation/maintenance, making it more suitable for repeated measurements. Measurements of S. polyrhiza root length may be suitable for assessment when copper and chromium in municipal and industrial wastewater exceed the environmentally permissible levels.

Metal Toxicity across Different Thallus Sections of the Green Macroalga, Ulva australis.

We aimed to identify functional differences between different sections of the thallus of Ulva australis and develop tissue-endpoint combinations to assess the toxicity of six metals (i.e., Ag, As, Cd, Cr, Cu, and Ni). EC50 values for these metals in three sections of the thallus of Ulva were obtained for multiple endpoints: relative growth rate (RGR), chlorophyll a fluorescence, pigment contents, and the expression of the photosynthesis-related gene, rbcL. The responses of the endpoints varied across the respective thallus sections; overall, the most toxic metals were Ag and Cu. These endpoints were the best for evaluating metal toxicity: ETRmax of the middle thallus sections for Ag toxicity; RGR of the middle thallus section for As and Cd; ETRmax of the marginal thallus section for Cr; Chl b contents of the marginal thallus section for Cu; RGR of the basal thallus section for Ni. The EC50 values for the inhibition of ETRmax in middle (0.06 mg∙L-1) and Chl b in the marginal thallus sections (0.06 mg∙L-1) were all lower than those of the quality standard for wastewater discharge values of Ag and Cu in Republic of Korea and the US, pointing to the suitability of U. australis-based endpoints for risk assessment.

Plasmonic Nanodomains Decorated on Two-Dimensional Oxide Semiconductors for Photonic-Assisted CO2 Conversion.

Plasmonic nanostructures ensure the reception and harvesting of visible lights for novel photonic applications. In this area, plasmonic crystalline nanodomains decorated on the surface of two-dimensional (2D) semiconductor materials represent a new class of hybrid nanostructures. These plasmonic nanodomains activate supplementary mechanisms at material heterointerfaces, enabling the transfer of photogenerated charge carriers from plasmonic antennae into adjacent 2D semiconductors and therefore activate a wide range of visible-light assisted applications. Here, the controlled growth of crystalline plasmonic nanodomains on 2D Ga2O3 nanosheets was achieved by sonochemical-assisted synthesis. In this technique, Ag and Se nanodomains grew on 2D surface oxide films of gallium-based alloy. The multiple contribution of plasmonic nanodomains enabled the visible-light-assisted hot-electron generation at 2D plasmonic hybrid interfaces, and therefore considerably altered the photonic properties of the 2D Ga2O3 nanosheets. Specifically, the multiple contribution of semiconductor-plasmonic hybrid 2D heterointerfaces enabled efficient CO2 conversion through combined photocatalysis and triboelectric-activated catalysis. The solar-powered acoustic-activated conversion approach of the present study enabled us to achieve the CO2 conversion efficiency of more than 94% in the reaction chambers containing 2D Ga2O3-Ag nanosheets.

Qualitative and quantitative assessment of diatom deformities and protoplasmic condition under metal and metalloid stress.

Metals and metalloids are toxic, persistent, and non-biodegradable and can be biomagnified (e.g., Hg), and therefore pose a serious threat to the algal flora of aquatic ecosystems. This laboratory study tested the effects of metals (Zn, Fe, and Hg) and a metalloid (As) on the cell wall morphology and protoplasmic content of living cells of six widespread diatom genera over 28 days. Diatoms exposed to Zn and Fe had a higher frequency of deformed diatom frustules (> 1%) compared to the As, Hg, and control treatments (< 1%). Deformities in the valve outline and striae were found in all treatments, including the control, whereas deformed raphes and more than one type of deformity were more prevalent under Zn and Hg stress. The order of toxicity is as follows: Zn > Fe > Hg≈As. Deformities were more frequent in Achnanthes and Diploneis (adnate forms) than in the motile genera of Nitzschia and Navicula. The correlation between the % healthy diatoms and % deformities in all six genera showed a negative relationship with the integrity of protoplasmic content (i.e., greater alteration in protoplasmic content was associated with greater frustule deformation). We conclude that diatom deformities can be a good indicator of metal and metalloid stress in waterbodies and are very useful in the rapid biomonitoring of aquatic ecosystems.

Microwave assisted and conventional hydrothermal treatment of waste seaweed: Comparison of hydrochar properties and energy efficiency.

Waste seaweed is a valuable source for converting into value-added carbon materials. In this study, the production of hydrochar from waste seaweed was optimized for hydrothermal carbonization in a microwave process. The produced hydrochar was compared with hydrochar synthesized by the regular process using a conventional heating oven. The results show that hydrochar produced with a holding time of 1 h by microwave heating has similar properties to the hydrochar produced in a conventionally heated oven for 4 h (200 °C and water/biomass ratio 5): carbon mass fraction (52.4 ± 3.9 %), methylene blue adsorption capacity (40.2 ± 0.2 mg g-1) and similar observations on surface functional groups and thermal stability were made between hydrochars produced by both methods. The analysis of energy consumption showed microwave assisted carbonization consume higher energy in compare to conventional oven. The present results suggest that hydrochar made from waste seaweed and using the microwave technique could be an energy-saving technology for producing hydrochar with similar specifications to hydrochar produced by conventional heating methods.

Evaluating ecotoxicological assays for comprehensive risk assessment of toxic metals present in industrial wastewaters in the Republic of Korea.

Toxicity tests represent a rapid, user-friendly and cost-effective means to assess the impact of wastewater quality on aquatic ecosystems. There are not many cases where wastewater management standards are set based on various bio-based ecotoxicity values. Here, we tested a novel multitaxon approach to compare standard water quality indices to toxicity metrics obtained from ecotoxicity tests, conducted using aquatic organisms representing several trophic levels (Aliivibrio, Ulva, Daphnia, and Lemna), for 99 industrial wastewater samples from South Korea. For five wastewater samples, the concentrations of Se, Zn, or Ni exceeded the permissible limits (1, 5, and 3 mg L-1, respectively). All the four physiochemical water quality indices tested were positively correlated with Se and Pb concentrations. The toxicity unit (TU) scores indicated a declining sensitivity to pollutants, in the order Lemna (2.87) >Daphnia (2.24) >Aliivibrio (1.78) >Ulva (1.42). Significant correlations were observed between (1) Cd and Ni, and Aliivibrio, (2) Cu and Daphnia, (3) Cd, Cu, Zn, and Cr and Lemna, and (4) Cu, Zn, and Ni and Ulva. Daphnia-Lemna and Lemna-Ulva were found to be good indicators of ecologically harmful Se and Ni contents in wastewater, respectively. We suggest that regulatory thresholds based on these bioassays should be set at TU = 1 for all the species or at TU = 1 for Aliivibrio and Ulva and TU = 2 for Daphnia and Lemna, if the number of companies whose wastewater discharge exceeds the allowable TU levels is <1 % or 5 % of the total number of industries, respectively. Taken together, these findings could help in establishing a rapid, ecologically relevant wastewater quality assessment system that would be useful for developing strategies to protect aquatic ecosystems.

IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction.

BACKGROUND: Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. RESULTS: Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. CONCLUSIONS: We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology.

A Ten-Minute Bioassay to Test Metal Toxicity with the Freshwater Flagellate Euglena agilis.

A chemical analysis of water quality cannot detect some toxicants due to time constraints, high costs, and limited interactions for detection. Bioassays would offer a complementary means to assess pollution levels in water. Euglena is a flagellate green alga and an excellent system for toxicity testing thanks to its ease of culture, rapid growth, and quick response to environmental stresses. Herein, we examined the sensitivity of E. agilis to seven heavy metals by analyzing six end-point parameters: motility, velocity, cell compactness, upward swimming, r-value, and alignment. Notably, the velocity of E. agilis was most sensitive to cadmium (96.28 mg·L-1), copper (6.51 mg·L-1), manganese (103.28 mg·L-1), lead (78.04 mg·L-1), and zinc (101.90 mg·L-1), while r-values were most sensitive to arsenic (12.84 mg·L-1) and mercury (4.26 mg·L-1). In this study, velocity and r-values are presented as useful biomarkers for the assessment of metal toxicity in Euglena. The metals As, Cd, Cu, and Pb were suitable for this test. The advantages of the ecotoxicity test are its rapidity: It takes 10 min to obtain results, as opposed to the typical 3-4 d of exposure time with intensive labor. Moreover, this test can be performed at room temperature under dark conditions.

Plasma Membrane Localization of CD36 Requires Vimentin Phosphorylation; A Mechanism by Which Macrophage Vimentin Promotes Atherosclerosis.

Vimentin is a type III intermediate filament protein expressed in cells of mesenchymal origin. Vimentin has been thought to function mainly as a structural protein and roles of vimentin in other cellular processes have not been extensively studied. Our current study aims to reveal functions of vimentin in macrophage foam cell formation, the critical stage of atherosclerosis. We demonstrated that vimentin null (Vim -/ - ) mouse peritoneal macrophages take up less oxidized LDL (oxLDL) than vimentin wild type (Vim +/+) macrophages. Despite less uptake of oxLDL in Vim -/ - macrophages, Vim +/+ and Vim -/ - macrophages did not show difference in expression of CD36 known to mediate oxLDL uptake. However, CD36 localized in plasma membrane was 50% less in Vim -/ - macrophages than in Vim +/+ macrophages. OxLDL/CD36 interaction induced protein kinase A (PKA)-mediated vimentin (Ser72) phosphorylation. Cd36 -/ - macrophages did not exhibit vimentin phosphorylation (Ser72) in response to oxLDL. Experiments using phospho-mimetic mutation of vimentin revealed that macrophages with aspartate-substituted vimentin (V72D) showed more oxLDL uptake and membrane CD36. LDL receptor null (Ldlr -/ - ) mice reconstituted with Vim -/ - bone marrow fed a western diet for 15 weeks showed 43% less atherosclerotic lesion formation than Ldlr -/ - mice with Vim +/+ bone marrow. In addition, Apoe -/ -Vim- / - (double null) mice fed a western diet for 15 weeks also showed 57% less atherosclerotic lesion formation than Apoe -/ - and Vim +/+mice. We concluded that oxLDL via CD36 induces PKA-mediated phosphorylation of vimentin (Ser72) and phosphorylated vimentin (Ser72) directs CD36 trafficking to plasma membrane in macrophages. This study reveals a function of vimentin in CD36 trafficking and macrophage foam cell formation and may guide to establish a new strategy for the treatment of atherosclerosis.

Commercial Potential of the Cyanobacterium Arthrospira maxima: Physiological and Biochemical Traits and the Purification of Phycocyanin.

Arthrospira maxima is a natural source of fine chemicals for multiple biotechnological applications. We determined the optimal environmental conditions for A. maxima by measuring its relative growth rate (RGR), pigment yield, and photosynthetic performance under different pH and temperature conditions. RGR was highest at pH 7-9 and 30 °C. Chlorophyll a, phycocyanin, maximal quantum yield (Fv/Fm), relative maximal electron transport rate (rETRmax), and effective quantum yield (ΦPSII) were highest at pH 7-8 and 25 °C. Interestingly, phycoerythrin and allophycocyanin content was highest at 15 °C, which may be the lowest optimum temperature reported for phycobiliprotein production in the Arthrospira species. A threestep purification of phycocyanin (PC) by ultrafiltration, ion-exchange chromatography, and gel filtration resulted in a 97.6% purity of PC.

Assessment of Various Toxicity Endpoints in Duckweed (Lemna minor) at the Physiological, Biochemical, and Molecular Levels as a Measure of Diuron Stress.

The common, broad-spectrum herbicide diuron poses some risks to the environment due to its long persistence and high toxicity. Therefore, the effective monitoring of diuron residues will inform efforts to assess its impacts on ecosystems. In this study, we evaluated the toxicity targets of diuron in the model aquatic macrophyte Lemna minor at the physiological (growth and photosynthetic efficiency), biochemical (pigment biosynthesis and reactive oxygen species (ROS) levels), and molecular (rbcL transcript) levels. The toxicity of diuron was detectable after 48 h of exposure and the order of sensitivity of toxicity endpoints was gene transcription > maximum electron transport rate (ETRmax) > non-photochemical quenching (NPQ) > maximum quantum yield (Fv/Fm) > ROS > fresh weight > chlorophyll b > chlorophyll a > total frond area > carotenoids. Under diuron stress, pigment, ROS, and gene transcript levels increased while frond area, fresh weight, and photosynthesis (Fv/Fm and ETRmax) gradually decreased with the increasing duration of exposure. Notably, ROS levels, Fv/Fm, frond area, and fresh weight were highly correlated with diuron concentration. The growth endpoints (frond area and fresh weight) showed a strong negative correlation with ROS levels and a positive correlation with Fv/Fm and ETRmax. These findings shed light on the relative sensitivity of different endpoints for the assessment of diuron toxicity.

Understanding Metabolic Regulation Between Host and Pathogens: New Opportunities for the Development of Improved Therapeutic Strategies Against Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (Mtb) causes chronic granulomatous lung disease in humans. Recently, novel strategies such as host-directed therapeutics and adjunctive therapies that enhance the effect of existing antibiotics have emerged to better control Mtb infection. Recent advances in understanding the metabolic interplay between host immune cells and pathogens have provided new insights into how their interactions ultimately influence disease outcomes and antibiotic-treatment efficacy. In this review, we describe how metabolic cascades in immune environments and relevant metabolites produced from immune cells during Mtb infection play critical roles in the progression of diseases and induction of anti-Mtb protective immunity. In addition, we introduce how metabolic alterations in Mtb itself can lead to the development of persister cells that are resistant to host immunity and can eventually evade antibiotic attacks. Further understanding of the metabolic link between host cells and Mtb may contribute to not only the prevention of Mtb persister development but also the optimization of host anti-Mtb immunity together with enhanced efficacy of existing antibiotics. Overall, this review highlights novel approaches to improve and develop host-mediated therapeutic strategies against Mtb infection by restoring and switching pathogen-favoring metabolic conditions with host-favoring conditions.

Interlaboratory Validation of Toxicity Testing Using the Duckweed Lemna minor Root-Regrowth Test.

The common duckweed (Lemna minor), a freshwater monocot that floats on the surfaces of slow-moving streams and ponds, is commonly used in toxicity testing. The novel Lemna root- regrowth test is a toxicity test performed in replicate test vessels (24-well plates), each containing 3 mL test solution and a 2-3 frond colony. Prior to exposure, roots are excised from the plant, and newly developed roots are measured after 3 days of regrowth. Compared to the three internationally standardized methods, this bioassay is faster (72 h), simpler, more convenient (requiring only a 3-mL) and cheaper. The sensitivity of root regrowth to 3,5-dichlorophenol was statistically the same as using the conventional ISO test method. The results of interlaboratory comparison tests conducted by 10 international institutes showed 21.3% repeatability and 27.2% reproducibility for CuSO4 and 21.28% repeatability and 18.6% reproducibility for wastewater. These validity criteria are well within the generally accepted levels of <30% to 40%, confirming that this test method is acceptable as a standardized biological test and can be used as a regulatory tool. The Lemna root regrowth test complements the lengthier conventional protocols and is suitable for rapid screening of wastewater and priority substances spikes in natural waters.

Rapid analytical approach for bioprofiling compounds with radical scavenging and antimicrobial activities from seaweeds.

Brown seaweeds are traditionally used as food in Asian countries, and they are a valuable source of bioactive compounds. Herein, a novel high-throughput methodological approach was developed for the tracing of compounds with radical scavenging and antimicrobial activities in Saccharina japonica and Undaria pinnatifida methanol extracts. The seaweed metabolites were separated by a novel high-performance thin-layer chromatography method, the bioactive bands were identified by bioautography assays. The bioactive compounds were characterized with ultra-high-performance liquid chromatography coupled with linear trap quadrupole tandem mass spectrometry. Stearidonic, eicosapentaenoic, and arachidonic acids were identified as major components having radical scavenging and antimicrobial activities. The suggested method provides a fast identification and quantification of bioactive compounds in multicomponent biological samples.