Application of ionizing radiation as an elicitor to enhance the growth and metabolic activities in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a eukaryotic, unicellular photosynthetic organism and a potential algal platform for producing biomass and recombinant proteins for industrial use. Ionizing radiation is a potent genotoxic and mutagenic agent used for algal mutation breeding that induces various DNA damage and repair responses. In this study, however, we explored the counterintuitive bioeffects of ionizing radiation, such as X- and γ-rays, and its potential as an elicitor to facilitate batch or fed-batch cultivation of Chlamydomonas cells. A certain dose range of X- and γ-rays was shown to stimulate the growth and metabolite production of Chlamydomonas cells. X- or γ-irradiation with relatively low doses below 10 Gy substantially increased chlorophyll, protein, starch, and lipid content as well as growth and photosynthetic activity in Chlamydomonas cells without inducing apoptotic cell death. Transcriptome analysis demonstrated the radiation-induced changes in DNA damage response (DDR) and various metabolic pathways with the dose-dependent expression of some DDR genes, such as CrRPA30, CrFEN1, CrKU, CrRAD51, CrOASTL2, CrGST2, and CrRPA70A. However, the overall transcriptomic changes were not causally associated with growth stimulation and/or enhanced metabolic activities. Nevertheless, the radiation-induced growth stimulation was strongly enhanced by repetitive X-irradiation and/or subsequent cultivation with an inorganic carbon source, i.e., NaHCO3, but was significantly inhibited by treatment of ascorbic acid, a scavenger of reactive oxygen species (ROS). The optimal dose range of X-irradiation for growth stimulation differed by genotype and radiation sensitivity. Here, we suggest that ionizing radiation within a certain dose range determined by genotype-dependent radiation sensitivity could induce growth stimulation and enhance metabolic activities, including photosynthesis, chlorophyll, protein, starch, and lipid synthesis in Chlamydomonas cells via ROS signaling. The counterintuitive benefits of a genotoxic and abiotic stress factor, i.e., ionizing radiation, in a unicellular algal organism, i.e., Chlamydomonas, may be explained by epigenetic stress memory or priming effects associated with ROS-mediated metabolic remodeling.

Application of Gamma Ray-Responsive Genes for Transcriptome-Based Phytodosimetry in Rice.

Transcriptome-based dose-response curves were recently applied to the phytodosimetry of gamma radiation in a dicot plant, Arabidopsis thaliana, as an alternative biological assessment of genotoxicity using DNA damage response (DDR) genes. In the present study, we characterized gamma ray-responsive marker genes for transcriptome-based phytodosimetry in a monocot plant, rice (Oryza sativa L.), and compared different phytodosimetry models between rice and Arabidopsis using gamma-H2AX, comet, and quantitative transcriptomic assays. The transcriptome-based dose-response curves of four marker genes (OsGRG, OsMutS, OsRAD51, and OsRPA1) were reliably fitted to quadratic or exponential decay equations (r2 > 0.99). However, the single or integrated dose-response curves of these genes were distinctive from the conventional models obtained by the gamma-H2AX or comet assays. In comparison, rice displayed a higher dose-dependency in the comet signal and OsRAD51 transcription, while the gamma-H2AX induction was more dose-dependent in Arabidopsis. The dose-dependent transcriptions of the selected gamma-ray-inducible marker genes, including OsGRG, OsMutS, OsRAD51, and OsRPA1 in rice and AtGRG, AtPARP1, AtRAD51, and AtRPA1E in Arabidopsis, were maintained similarly at different vegetative stages. These results suggested that the transcriptome-based phytodosimetry model should be further corrected with conventional genotoxicity- or DDR-based models despite the high reliability or dose-dependency of the model. In addition, the relative weighting of each gene in the integrated transcriptome-based dose-response model using multiple genes needs to be considered based on the trend and amplitude of the transcriptional change.

Genetic Impairment of Cellulose Biosynthesis Increases Cell Wall Fragility and Improves Lipid Extractability from Oleaginous Alga Nannochloropsis salina.

In microalgae, photosynthesis provides energy and sugar phosphates for the biosynthesis of storage and structural carbohydrates, lipids, and nitrogenous proteins. The oleaginous alga Nannochloropsis salina does not preferentially partition photoassimilates among cellulose, chrysolaminarin, and lipids in response to nitrogenous nutrient deprivation. In the present study, we investigated whether genetic impairment of the cellulose synthase gene (CesA) expression would lead to protein accumulation without the accumulation of storage C polymers in N. salina. Three cesA mutants were generated by the CRISPR/Cas9 approach. Cell wall thickness and cellulose content were reduced in the cesA1 mutant, but not in cesA2 or cesA4 cells. CesA1 mutation resulted in a reduction of chrysolaminarin and neutral lipid contents, by 66.3% and 37.1%, respectively, but increased the soluble protein content by 1.8-fold. Further, N. salina cells with a thinned cell wall were susceptible to mechanical stress, resulting in a 1.7-fold enhancement of lipid extractability. Taken together, the previous and current studies strongly suggest the presence of a controlling mechanism that regulates photoassimilate partitioning toward C and N metabolic pathways as well as the cellulose metabolism as a potential target for cost-effective microalgal cell disruption and as a useful protein production platform.

Comparison of Ultrasound-Guided Versus Landmark-Based Corticosteroid Injection for Carpal Tunnel Syndrome: A Prospective Randomized Trial.

PURPOSE: Although a local corticosteroid injection for carpal tunnel syndrome (CTS) is frequently performed by palpation using anatomical landmarks, ultrasound (US) allows physicians to visualize and confirm placement of the injectate close to the median nerve, possibly improving the efficacy of the injection. The aim of this study was to compare the effectiveness and complications of US-guided steroid injections with landmark-based injections for CTS. METHODS: A total of 102 patients with CTS were randomized into 2 groups: landmark-based injection and US-guided injection. The response to treatment, including grip strength and the Boston Carpal Tunnel Questionnaires (BCTQ) was assessed at baseline and at 4, 12, and 24 weeks after the injection. RESULTS: The BCTQ symptom and function scores were similar in the 2 groups throughout the 24-week follow-up period, with the exception of significantly lower (better) symptom scores at 4-week follow-up in the US-guided injection group. The grip strength was similar in the 2 groups throughout the 24-week follow-up period. After 24 weeks, 12 patients (24%) in the landmark-based injection group and 9 patients (18%) in the US-guided injection group had undergone carpal tunnel surgery. Symptoms of median nerve irritation were more likely to occur in patients with landmark-based injections (14%) than in those with US-guided injection (2%). CONCLUSIONS: A US-guided steroid injection for CTS produces pain and functional results similar to those of landmark-based injection. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic I.

Elevated Inorganic Carbon Concentrating Mechanism Confers Tolerance to High Light in an Arctic Chlorella sp. ArM0029B.

Microalgae and higher plants employ an inorganic carbon (Ci) concentrating mechanism (CCM) to increase CO2 availability to Rubisco. Operation of the CCM should enhance the activity of the Calvin cycle, which could act as an electron sink for electrons generated by photosynthesis, and lower the redox status of photosynthetic electron transport chains. In this study, a hypothesis that microalgal cells with fully operating CCM are less likely to be photodamaged was tested by comparing a Chlorella mutant with its wild type (WT). The mutant acquired by screening gamma-ray-induced mutant libraries of Chlorella sp. ArM0029B exhibited constitutively active CCM (CAC) even in the presence of additional Ci sources under mixotrophic growth conditions. In comparison to the WT alga, the mutant named to constitutively active CCM1 (CAC1) showed more transcript levels for genes coding proteins related to CCM such as Ci transporters and carbonic anhydrases (CA), and greater levels of intracellular Ci content and CA activity regardless of whether growth is limited by light or not. Under photoinhibitory conditions, CAC1 mutant showed faster growth than WT cells with more PSII reaction center core component D1 protein (encoded by psbA), higher photochemical efficiency as estimated by the chlorophyll fluorescence parameter (Fv/Fm), and fewer reactive oxygen species (ROS). Interestingly, high light (HL)-induced increase in ROS contents in WT cells was significantly inhibited by bicarbonate supplementation. It is concluded that constitutive operation of CCM endows Chlorella cells with resistance to HL partly by reducing the endogenous generation of ROS. These results will provide useful information on the interaction between CCM expression, ROS production, and photodamage in Chlorella and related microalgae.

Transcriptional Regulation of Cellulose Biosynthesis during the Early Phase of Nitrogen Deprivation in Nannochloropsis salina.

Microalgal photosynthesis provides energy and carbon-containing precursors for the biosynthesis of storage carbohydrates such as starch, chrysolaminarin, lipids, and cell wall components. Under mild nitrogen deficiency (N-), some Nannochloropsis species accumulate lipid by augmenting cytosolic fatty acid biosynthesis with a temporary increase in laminarin. Accordingly, biosynthesis of the cellulose-rich cell wall should change in response to N- stress because this biosynthetic pathway begins with utilisation of the hexose phosphate pool supplied from photosynthesis. However, few studies have characterised microalgal cell wall metabolism, including oleaginous Nannochloropsis sp. microalgae subjected to nitrogen deficiency. Here, we investigated N-induced changes in cellulose biosynthesis in N. salina. We observed that N- induced cell wall thickening, concurrently increased the transcript levels of genes coding for UDPG pyrophosphorylase and cellulose synthases, and increased cellulose content. Nannochloropsis salina cells with thickened cell wall were more susceptible to mechanical stress such as bead-beating and sonication, implicating cellulose metabolism as a potential target for cost-effective microalgal cell disruption.

CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii.

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms.

Threonine 286 of fatty acid desaturase 7 is essential for ω-3 fatty acid desaturation in the green microalga Chlamydomonas reinhardtii.

Omega-3 fatty acid desaturases catalyze the conversion of dienoic fatty acids (C18:2 and C16:2) into trienoic fatty acids (C18:3 and C16:3), accounting for more than 50% of the total fatty acids in higher plants and the green microalga Chlamydomonas reinhardtii. Here, we describe a Thr residue located in the fourth transmembrane domain of fatty acid desaturase 7 (FAD7) that is essential for the biosynthesis of ω-3 fatty acids in C. reinhardtii. The ω-3 fatty acid deficiency in strain CC-620, which contains a putative missense mutation at Thr286 of CrFAD7, was recovered by the overexpression of CC-125 CrFAD7. A Ser substitution in position 286 was able to partially complement the phenotype of the ω-3 fatty acid deficiency, but other substitution variants, such as Tyr, His, Cys, and Gly, failed to do so. Prediction of the phosphorylation target site revealed that Thr286 may be phosphorylated. Analysis of the structural conformation of CC-620 CrFAD7 via topology prediction (and bends in the helix) shows that this missense mutation may collapse the catalytic structure of CrFAD7. Taken together, this study suggests that Thr286 is essential for the maintaining the catalytic structure of CrFAD7.

A new Arctic Chlorella species for biodiesel production.

Microalgae are a potential resource for biodiesel production. A green alga, Chlorella sp., was isolated from Arctic sea ice, which was named ArM0029B. These algae displayed faster growth at a wide temperature range of 4-32°C compared to Chlorella vulgaris. ArM0029B also accumulated high levels of total fatty acids under nitrogen starvation conditions, reaching 39% of dry cell weight, with the proportion of oleic acid (18:1) and linoleic acid (18:2) reaching 54% of total fatty acids. Taken together, these results indicate that the newly identified Chlorella species, ArM0029B, is a promising candidate for biodiesel production.