Dihydroartemisinin inhibits HNSCC invasion and migration by controlling miR-195-5p expression.

Objectives: Dihydroartemisinin (DHA), an artemisinin derivative extracted from the traditional Chinese medicinal herb Artemisia annua, has the potential to suppress head and neck squamous cell carcinoma (HNSCC) progression. However, the mechanisms underlying these effects remain unclear. Therefore, we aimed to examine the mechanisms underlying the effects of DHA on tumor invasion and migration. Methods: Human HNSCC cell lines CAL-27 and FaDu were exposed to varying DHA concentrations (0, 5, 20, and 80 µM) for 24 h. Cell proliferation, invasion, and migration were assessed using CCK8, transwell, and wound-healing assays, respectively. Quantitative real-time PCR, western blotting, and immunofluorescence were used to assess the expression levels of the target genes and proteins. Results: DHA suppressed the invasion and migration of CAL-27 and FaDu cells. Additionally, miR-195-5p suppressed the invasion and migration of HNSCC cells. This study revealed significant differences in the expression of miR-195-5p and TENM2 between clinical samples and multiple public databases. DHA treatment and miR-195-5p overexpression significantly reduced TENM2 expression in HNSCC cells, which suggested that miR-195-5p overexpression enhanced the inhibitory effect of DHA on TENM2. Conclusions: This study provides the first evidence that DHA inhibits cell invasion and migration by regulating the miR-195-5p/TENM2 axis in HNSCC cells, suggesting it as a potentially effective treatment strategy for HNSCC.

A Proactive Intervention Study in Metabolic Syndrome High-Risk Populations Using Phenome-Based Actionable P4 Medicine Strategy.

The integration of predictive, preventive, personalized, and participatory (P4) healthcare advocates proactive intervention, including dietary supplements and lifestyle interventions for chronic disease. Personal profiles include deep phenotypic data and genetic information, which are associated with chronic diseases, can guide proactive intervention. However, little is known about how to design an appropriate intervention mode to precisely intervene with personalized phenome-based data. Here, we report the results of a 3-month study on 350 individuals with metabolic syndrome high-risk that we named the Pioneer 350 Wellness project (P350). We examined: (1) longitudinal (two times) phenotypes covering blood lipids, blood glucose, homocysteine (HCY), and vitamin D3 (VD3), and (2) polymorphism of genes related to folic acid metabolism. Based on personalized data and questionnaires including demographics, diet and exercise habits information, coaches identified 'actionable possibilities', which combined exercise, diet, and dietary supplements. After a 3-month proactive intervention, two-thirds of the phenotypic markers were significantly improved in the P350 cohort. Specifically, we found that dietary supplements and lifestyle interventions have different effects on phenotypic improvement. For example, dietary supplements can result in a rapid recovery of abnormal HCY and VD3 levels, while lifestyle interventions are more suitable for those with high body mass index (BMI), but almost do not help the recovery of HCY. Furthermore, although people who implemented only one of the exercise or diet interventions also benefited, the effect was not as good as the combined exercise and diet interventions. In a subgroup of 226 people, we examined the association between the polymorphism of genes related to folic acid metabolism and the benefits of folate supplementation to restore a normal HCY level. We found people with folic acid metabolism deficiency genes are more likely to benefit from folate supplementation to restore a normal HCY level. Overall, these results suggest: (1) phenome-based data can guide the formulation of more precise and comprehensive interventions, and (2) genetic polymorphism impacts clinical responses to interventions. Notably, we provide a proactive intervention example that is operable in daily life, allowing people with different phenome-based data to design the appropriate intervention protocol including dietary supplements and lifestyle interventions. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00115-z.

Development and validation of a nomogram for predicting poor operative visibility during FESS in Chinese adult patients with CRS.

Objective: The purpose of this study is to develop and evaluate a nomogram that is capable of predicting poor operative visibility during functional endoscopic sinus surgery. Method: To identify potential risk factors, patients with chronic rhinosinusitis who underwent functional endoscopic sinus surgery (FESS) between January 2019 and December 2022 were selected from our hospital's electronic medical record system. Data on general patient information, clinical manifestations, clotting-related test indices, Lund-Machay score of sinuses CT scanning, Lund-kennedy score of nasal endoscopies, anesthesia methods, intraoperative blood pressure and heart rate, and Boezaart bleeding score were collected. Minimum absolute convergence and selection operator (LASSO) regression, as well as multivariate logistic regression, were used to determine the risk factors. A nomogram was developed in order to predict poor operating visibility during FESS, and its performance was evaluated utilizing both the training and verification datasets via various measures including receiver operating characteristic (ROC) curve analysis, area under the curve (AUC), Hosmer-Lemeshow goodness-of-fit test, calibration curve, and decision curve analysis. Results: Of the 369 patients who met the inclusion criteria, 88 of them exhibited POV during FESS. By deploying LASSO and multivariate logistic regression analyses, six risk factors were identified and used to construct a nomogram for predicting POV during FESS. These factors include prothrombin time (PT), prothrombin activity (PTA), Lund-Mackay score (LMS), Lund-Kennedy score (LKS), anesthetic method, and intraoperative hypertension. The AUC of the training set was found to be 0.820 while that of the verification set was 0.852. The Hosmer-Lemeshow goodness-of-fit test and calibration curve analysis revealed good consistency between predicted and actual probabilities. Also, the decision curve demonstrated that the nomogram had a high degree of clinical usefulness and net benefit. Conclusion: The constructed nomogram has a strong ability to predict the poor intraoperative field in patients with chronic rhinosinusitis, which can help preoperative judgment of high-risk patients and provide evidence for perioperative management and preoperative plan formulation.

Impact of environmental factors changes induced by marine heatwaves and heavy precipitation on antibiotic toxicity to Isochrysis galbana: Implications for climate change adaptation.

Isochrysis galbana, a crucial primary producer and food source in aquatic ecosystems, faces increasing challenges from climate change and emerging contaminants like antibiotics. This study investigates the combined effects of sudden temperature increase (representing marine heatwaves) and rapid salinity change (representing extreme precipitation events) on the toxicity of tetracycline (TC) and oxytetracycline (OTC) to I. galbana. Short-term experiments reveal heightened antibiotic toxicity at 31 °C or salinities of 18 PSU, surpassing algal tolerance limits. Long-term tests show decreased inhibition of algal growth on day 9, indicating algal adaptation to the environment. Analyses of photosynthesis II efficiency, pigment content, and macromolecular composition support this, suggesting adaptation mechanism activation. While algae acclimate to the environment during long-term antibiotic exposure, extreme weather conditions may compromise this adaptation. These findings have implications for managing antibiotics in aquatic environments under climate change.

Expansion of human megakaryocyte-biased hematopoietic stem cells by biomimetic Microniche.

Limited numbers of available hematopoietic stem cells (HSCs) limit the widespread use of HSC-based therapies. Expansion systems for functional heterogenous HSCs remain to be optimized. Here, we present a convenient strategy for human HSC expansion based on a biomimetic Microniche. After demonstrating the expansion of HSC from different sources, we find that our Microniche-based system expands the therapeutically attractive megakaryocyte-biased HSC. We demonstrate scalable HSC expansion by applying this strategy in a stirred bioreactor. Moreover, we identify that the functional human megakaryocyte-biased HSCs are enriched in the CD34+CD38-CD45RA-CD90+CD49f lowCD62L-CD133+ subpopulation. Specifically, the expansion of megakaryocyte-biased HSCs is supported by a biomimetic niche-like microenvironment, which generates a suitable cytokine milieu and supplies the appropriate physical scaffolding. Thus, beyond clarifying the existence and immuno-phenotype of human megakaryocyte-biased HSC, our study demonstrates a flexible human HSC expansion strategy that could help realize the strong clinical promise of HSC-based therapies.

New Insights into Xanthophylls and Lipidomic Profile Changes Induced by Glucose Supplementation in the Marine Diatom Nitzschia laevis.

Nitzschia laevis is a candidate microorganism for bioactive compounds (fucoxanthin and eicosapentaenoic acid (EPA)) production. In this study, the impacts of glucose-induced trophic transition on biomass, photosynthesis, pigments, and lipid profiles were examined. The specific growth rate was increased under glucose addition, achieved at 0.47 day-1 (0.26 ± 0.01 day-1 for the group without glucose in medium). However, the photosynthetic parameters and pigments including chlorophylls, fucoxanthin, and diatoxanthin were reduced. The net yield of EPA doubled under glucose addition, reaching 20.36 ± 1.22 mg/L in 4 days. In addition, the alteration in detailed lipid molecular species was demonstrated with a focus on EPA-enriched lipids. The effects of 2-deoxyglucose (2DG) indicated that glucose phosphorylation was involved in glucose-induced regulation. These findings provide novel data for guiding the application of this diatom strain in the functional food industries.

Efficacy and Safety of Anlotinib Combined with PD-1 Blockades for Patients with Previously Treated Epithelial Ovarian Cancer: A Retrospective Study.

Purpose: This study was to investigate the efficacy and safety of anlotinib combined with programmed cell death protein 1 (PD-1) blockades for patients with previously treated advanced epithelial ovarian cancer (EOC). Patients and Methods: Present study was designed as a retrospective study, a total of 32 patients with advanced EOC who progressed after at least two lines previously available standard therapy were included in this study. All the patients were administered with anlotinib combined with PD-1 blockades administration. Clinical activity was implemented and analyzed, which was assessed according to the change of target lesion by imaging evidence and all the subjects were followed up regularly. Safety profile were collected and documented during the treatment. Univariate analysis was carried out using log rank test and multivariate analysis were adjusted by Cox regression analysis. Results: The best overall response suggested that partial response was noted in 12 patients, stable disease was observed in 14 patients, progressive disease was found in 6 patients. Therefore, the objective response rate (ORR) of the 32 patients was 37.5% (95% CI: 21.1-56.3%), disease control rate (DCR) of the patients was 81.3% (95% CI: 63.6-92.8%). The median follow-up duration of this study was 17.5 months (follow-up range: 0.9-33.5 months). And the median PFS and OS of the 32-patient cohort was 6.8 months (95% CI: 2.64-10.96) and 18.5 months (95% CI: 14.08-22.92), respectively. The most common treatment-related adverse reactions were fatigue (68.8%), nausea and vomiting (56.3%), hypertension (50.0%) and diarrhea (40.6%). Multivariate Cox regression analysis for PFS indicated that ECOG performance status and FIGO stage were independent factors to predict PFS of patients with previously treated EOC. Conclusion: Anlotinib combined with PD-1 blockades demonstrated promising efficacy and tolerable safety profile for patients with previously treated advanced EOC preliminarily. The conclusion should be confirmed in more patients with advanced EOC subsequently.

Observation on the effect of periodontal treatment on patients with combined periodontal-pulpal lesions.

OBJECTIVE: To evaluate the effect of periodontal treatment on combined periodontal-pulpal lesions. METHODS: A total of 327 patients with periodontal-pulpal lesions (360 affected teeth) were selected, and all affected teeth were treated with a complete root canal, and assigned into group A (periodontal treatment group, 180 affected teeth) and group B (non-periodontal treatment group, 180 affected teeth). Group A received periodontal basic treatment for 2 weeks after the completion of root canal treatment; 6 weeks later, if there were still more than 5 mm periodontal pockets and bleeding after detection, flap treatment was performed. Group B received root canal treatment and supragingival scaling. Follow-up was conducted at 3, 6, 12 and 24 months after surgery by observing the periodontal depth (PD), alveolar bone resorption and tooth mobility (TM). RESULT: In group A, the PDs before operation and 2 years after operation were (5.966±1.877) mm and (5.133±1.935) mm, and the PD was significantly decreased. In group B, the PDs before operation and 2 years after operation were (5.533±1.856) mm and (6.167±1.927) mm, and the PD was increased. There was no statistical difference in preoperative TM between the two groups (P>0.05). Two years after operation, TM in group A was significantly lower than that in group B (P<0.05). In terms of X-ray performance, there was no significant change in alveolar bone resorption in group A two years after operation compared with that before operation (P>0.05); two years after operation, alveolar bone resorption in group B was significantly reduced compared with that before operation (P<0.05). CONCLUSION: Periodontal treatment is a promising technique for patients with combined periodontal-pulpal lesions.

Exome-Wide Association Study Identifies East Asian-Specific Missense Variant MTHFR C136T Influencing Homocysteine Levels in Chinese Populations RH: ExWAS of tHCY in a Chinese Population.

Plasma total homocysteine (tHCY) is a known risk factor of a wide range of complex diseases. No genome scans for tHCY have been conducted in East Asian populations. Here, we conducted an exome-wide association study (ExWAS) for tHCY in 5,175 individuals of Chinese Han origin, followed by a replication study in 668 Chinese individuals. The ExWAS identified two loci, 1p36.22 (lead single-nucleotide polymorphism (SNP) rs1801133, MTHFR C677T) and 16q24.3 (rs1126464, DPEP1), showing exome-wide significant association with tHCY (p < 5E-7); and both loci have been previously associated with tHCY in non-East Asian populations. Both SNPs were replicated in the replication study (p < 0.05). Conditioning on the genotype of C677T and rs1126464, we identified a novel East Asian-specific missense variant rs138189536 (C136T) of MTHFR (p = 6.53E-10), which was also significant in the replication study (p = 9.8E-3). The C136T and C677T variants affect tHCY in a compound heterozygote manner, where compound heterozygote and homozygote genotype carriers had on average 43.4% increased tHCY than had other genotypes. The frequency of the homozygote C677T genotype showed an inverse-U-shaped geospatial pattern globally with a pronounced frequency in northern China, which coincided with the high prevalence of hyperhomocysteinemia (HHCY) in northern China. A logistic regression model of HHCY status considering sex, age, and the genotypes of the three identified variants reached an area under the receiver operating characteristic curve (AUC) value of 0.74 in an independent validation cohort. These genetic observations provide new insights into the presence of multiple causal mutations at the MTHFR locus, highlight the role of genetics in HHCY epidemiology among different populations, and provide candidate loci for future functional studies.

Transcriptomics and Metabolomics Analyses Provide Novel Insights into Glucose-Induced Trophic Transition of the Marine Diatom Nitzschia laevis.

Diatoms have important ecological roles and are natural sources of bioactive compounds. Nitzschia laevis is a member of marine diatoms that accumulates high-value products including fucoxanthin and eicosapentaenoic acid (EPA). In this study, physiological data showed that comparing to autotrophic growth, mixotrophic cultivation with glucose supplementation led to a decrease of chlorophyll and fucoxanthin content in N. laevis, and an increase of biomass density and EPA yield. To further examine the metabolic barriers for fucoxanthin and EPA biosynthesis, comparative transcriptomic and metabolome analyses were conducted, with a focus on the genes related to carotenoids biosynthesis and fatty acid metabolism. The results indicated that phytoene desaturase (PDS) and zeta-carotene isomerase (ZISO) could be the rate-limiting enzymes in carotenoid biosynthesis. The transcription regulation of 3-ketoacyl-CoA synthase (KCS) and elongation of very long chain fatty acids protein (EVOVL) are important contributors associated with polyunsaturated fatty acids (PUFAs) accumulation. Furthermore, we also investigated the glucose-associated regulatory genes using weighted gene co-expression network analysis, and identified potential hub genes linked with cell cycle, carbohydrate metabolism, purine biosynthesis, and lipid metabolism. This study offers a high-quality transcriptome resource for N. laevis and provides a molecular framework for further metabolic engineering studies on fucoxanthin and EPA production.

Regulatory mechanism of miR-525-5p in over-invasion of trophoblast.

AIM: To investigate the mechanism of miRNA-525-5p (miR-525-5p) in regulating the invasion of trophoblast cells. METHODS: The expressions of miR-525-5p and Homeobox D10 (HOXD10) in pre-eclampsia (PE) and normal placentas were detected. Besides the expressions of miR-525-5p and HOXD10, the levels of Vimentin, N-cadherin and E-cadherin in human trophoblast (HTR)-8 cells were also measured after cell transfection. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) and Transwell assays assessed the proliferative and invasive capabilities of HTR-8 cells, respectively. Dual-luciferase reporter assay verified the targeting relationship between miR-525-5p and HOXD10. RESULTS: MiR-525-5p was lowly expressed and HOXD10 was highly expressed in PE placentas. MiR-525-5p inhibition or HOXD10 overexpression suppressed proliferation, invasion and epithelial-mesenchymal transition (EMT) of HTR-8 cells. MiR-525-5p overexpression or HOXD10 knockdown promoted proliferation, invasion and EMT of HTR-8 cells. HOXD10 was a downstream target of miR-525-5p. Inhibiting HOXD10 reversed the suppressive effects of miR-525-5p inhibition on proliferation, invasion and EMT of HTR-8 cells. CONCLUSION: MiR-525-5p mediates the invasion of trophoblast cells by regulating HOXD10, which provides new therapeutic targets for PE treatment.

Time­resolved transcriptome analysis during transitions of sulfur nutritional status provides insight into triacylglycerol (TAG) and astaxanthin accumulation in the green alga Chromochloris zofingiensis.

BACKGROUND: Chromochloris zofingiensis, an oleaginous microalga, is a promising feedstock for the co-production of triacylglycerol (TAG)-based biodiesel and the high-value product astaxanthin. To reveal the molecular mechanism of TAG and astaxanthin biosynthesis during transitions of sulfur nutritional status, namely sulfur-starvation (SS) and sulfur-replenishment (SR), the physiological responses and the transcriptomic dynamics of C. zofingiensis were examined. RESULTS: The results revealed a reversible TAG and astaxanthin accumulation under SS, which is correlated with the reduction of cell growth and protein content, indicating the reallocation of carbon. By correlating the data on the physiological and transcriptional responses to different sulfur nutritional status, a model for the underlying mechanism of TAG and astaxanthin accumulation in C. zofingiensis was postulated, which involved up-regulation of key genes including diacylglycerol acyltransferase (DGTT5) and beta-carotene ketolase (BKT1), increased energy and NADPH supply by elevating the tricarboxylic acid (TCA) cycle and the oxidative pentose phosphate (OPP) pathway, and the increased carbon precursors (pyruvate and acetyl-CoA) through central carbon metabolism. In addition, the net enhancement of the de novo biosynthesis of fatty acids and the re-direction of the terpenoid precursors toward the branch catalyzed by lycopene beta cyclase (LCYb) and BKT1 escalated the substrate availability for the biosynthesis of TAG and astaxanthin, respectively. CONCLUSIONS: In this study, the time-resolved transcriptional analysis of C. zofingiensis under SS and SR conditions was reported for the first time to elucidate the regulatory roles of key enzymes, including DGTT5, BKT1 and LCYb, in the underlying mechanisms of TAG and astaxanthin accumulation.

Improving glucose metabolism in the auditory cortex delays the aging of auditory function of guinea pig.

The glucose homeostasis is essential for brain function, and energy deficiency is a key feature of brain aging. We investigated whether improving glucose metabolism in the auditory cortex can delay the aging of auditory function of guinea pigs with age-related hearing loss (ARHL) by d-galactose. Auditory function was assessed by auditory brainstem response (ABR), glucose metabolism was detected by micro PET/CT, and the proteome were identified in auditory cortex by two-dimensional electrophoresis and matrix assisted laser desorption/ionization mass spectrometry. Glucose metabolism decreased in the auditory cortex of d-galactose group, and improving glucose metabolism can delay the aging of auditory function by upregulating seven metabolism-related proteins including ATP synthase subunit beta, triosephosphate isomerase, creatine kinase U-type, pyruvate dehydrogenase E1 component subunit beta, alpha-enolase, phosphoglycerate kinase, and tubulin beta-2A chain. These results suggest that the decrease of glucose metabolism in the auditory cortex may be an important role in the aging of auditory function, and improving glucose metabolism in the auditory cortex can delay the aging of auditory function of guinea pig with ARHL induced by d-galactose.

Astaxanthin Is Ketolated from Zeaxanthin Independent of Fatty Acid Synthesis in Chromochloris zofingiensis.

The biosynthesis of astaxanthin, a high-value keto-carotenoid with broad industrial applications, remains unambiguous in algae. Here, we dissected the astaxanthin biosynthetic pathway and the coordination between astaxanthin and triacylglycerol (TAG) biosynthesis in the emerging model alga Chromochloris zofingiensis In vivo and in vitro experiments demonstrated that astaxanthin, utilizing the methylerythritol phosphate pathway-derived isopentenyl diphosphate as the building block, was synthesized from ß-carotenoid ketolase-mediated ketolation of zeaxanthin rather than ß-carotenoid hydroxylase-mediated hydroxylation of canthaxanthin, thus leading to the buildup of astaxanthin and canthaxanthin as end products in C. zofingiensis The synthesized astaxanthin, stored in TAG-filled lipid droplets, was esterified mainly with the fatty acid C18:1, which was not catalyzed by any acyltransferase previously proposed. Astaxanthin accumulated in a well-coordinated manner with TAG, supported by the coordinated up-regulation of both biosynthetic pathways at the transcriptional level. Nevertheless, astaxanthin and TAG showed no interdependence: inhibition of de novo fatty acid biosynthesis severely attenuated TAG biosynthesis but promoted the accumulation of astaxanthin, particularly in the diester form, leading to a fivefold increase in the astaxanthin/TAG ratio; however, inhibition of astaxanthin biosynthesis showed little effect on TAG accumulation. Our data suggest that an increase in astaxanthin accumulation following inhibition of de novo fatty acid biosynthesis, which is not regulated at the transcriptional level, is likely derived from the conversion of other carotenoids rather than from a shunt of carbon flux from lipid biosynthesis. Combined, these findings further our understanding of astaxanthin biosynthesis and provide a feasible strategy for promoting astaxanthin content and purity in algae.

Harnessing C/N balance of Chromochloris zofingiensis to overcome the potential conflict in microalgal production.

Accumulation of high-value products in microalgae is not conducive with rapid cell growth, which is the potential conflict in microalgal production. Overcoming such conflict faces numerous challenges in comprehensively understanding cell behavior and metabolism. Here, we show a fully integrated interaction between cell behavior, carbon partitioning, carbon availability and path rate of central carbon metabolism, and have practically overcome the production conflict of Chromochloris zofingiensis. We demonstrate that elevated carbon availability and active path rate of precursors are determinants for product biosynthesis, and the former exhibits a superior potential. As protein content reaches a threshold value to confer survival advantages, carbon availability becomes the major limiting factor for product biosynthesis and cell reproduction. Based on integrated interaction, regulating the C/N balance by feeding carbon source under excess light increases content of high-value products without inhibiting cell growth. Our findings provide a new orientation to achieve great productivity improvements in microalgal production.

Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study.

BACKGROUND: Chromochloris zofingiensis, a freshwater alga capable of synthesizing both triacylglycerol (TAG) and astaxanthin, has been receiving increasing attention as a leading candidate producer. While the mechanism of oleaginousness and/or carotenogenesis has been studied under such induction conditions as nitrogen deprivation, high light and glucose feeding, it remains to be elucidated in response to salt stress, a condition critical for reducing freshwater footprint during algal production processes. RESULTS: Firstly, the effect of salt concentrations on growth, lipids and carotenoids was examined for C. zofingiensis, and 0.2 M NaCl demonstrated to be the optimal salt concentration for maximizing both TAG and astaxanthin production. Then, the time-resolved lipid and carotenoid profiles and comparative transcriptomes and metabolomes were generated in response to the optimized salt concentration for congruent analysis. A global response was triggered in C. zofingiensis allowing acclimation to salt stress, including photosynthesis impairment, ROS build-up, protein turnover, starch degradation, and TAG and astaxanthin accumulation. The lipid metabolism involved a set of stimulated biological pathways that contributed to carbon precursors, energy and reductant molecules, pushing and pulling power, and storage sink for TAG accumulation. On the other hand, salt stress suppressed lutein biosynthesis, stimulated astaxanthin biosynthesis (mainly via ketolation), yet had little effect on total carotenoid flux, leading to astaxanthin accumulation at the expense of lutein. Astaxanthin was predominantly esterified and accumulated in a well-coordinated manner with TAG, pointing to the presence of common regulators and potential communication for the two compounds. Furthermore, the comparison between salt stress and nitrogen deprivation conditions revealed distinctions in TAG and astaxanthin biosynthesis as well as critical genes with engineering potential. CONCLUSIONS: Our multi-omics data and integrated analysis shed light on the salt acclimation of C. zofingiensis and underlying mechanisms of TAG and astaxanthin biosynthesis, provide engineering implications into future trait improvements, and will benefit the development of this alga for production uses under saline environment, thus reducing the footprint of freshwater.

Functional Characterization of Long-Chain Acyl-CoA Synthetase Gene Family from the Oleaginous Alga Chromochloris zofingiensis.

Long-chain acyl-coenzyme A (CoA) synthetase (LACS) catalyzes the formation of acyl-CoAs from free fatty acids, which is pivotal for lipid metabolism. Here, we confirmed the presence of six CzLACS genes in Chromochloris zofingiensis. Functional complementation and in vitro enzymatic assay indicated that CzLACS2 through CzLACS5 rather than CzLACS1 or CzLACS6 are bona fide LACS enzymes and they have overlapping yet distinct substrate preference. The results of the subcellular colocalization experiment and different expression patterns under three triacylglycerol (TAG)-inducing conditions showed that CzLACS2 through CzLACS4 reside at endoplasmic reticulum (ER) and are involved in TAG biosynthesis, while CzLACS5 resides in peroxisome and participates in fatty acid ß-oxidation. The yeast one-hybrid assay using a library of 50 transcription factors (TFs) constructed in our study identified 12 TFs potentially involved in regulating the expression of CzLACSs. Moreover, heterologous expression of CzLACSs demonstrated their engineering potential for modulating TAG synthesis in yeast and algal cells.

Boost carbon availability and value in algal cell for economic deployment of biomass.

This study elucidated storage carbon metabolism in a dynamic manner through kinetic model, metabolomics and stable metabolic flux analysis. Results revealed nutrient uptake rate, carbon availability and synthetic path rate accounted for the integration of process-compatible products. The uptake rate could be enhanced by promoting carbohydrate accumulation, inducing high performance of tricarboxylic acid cycle and anaplerotic routes. Values of specific rate for lipid from kinetic model and synthetic path rate from metabolic flux analysis revealed that conversion of carbon sinks occupied a key position in increasing productivities of lipid and astaxanthin to 302.34 and 1.83 mg g-1 d-1, respectively. Additionally, economic estimation was applied to link cultivation factors with market scenario and demonstrated that regulating such carbon metabolism raised 30% increase of biomass value. This study therefore provided a new orientation to boost carbon efficiency that helped to engineer carbon flux from carbon source to targeted products precisely and rapidly.

Proteomics Analysis of Lipid Droplets from the Oleaginous Alga Chromochloris zofingiensis Reveals Novel Proteins for Lipid Metabolism.

Chromochloris zofingiensis represents an industrially relevant and unique green alga, given its capability of synthesizing triacylglycerol (TAG) and astaxanthin simultaneously for storage in lipid droplets (LDs). To further decipher lipid metabolism, the nitrogen deprivation (ND)-induced LDs from C. zofingiensis were isolated, purified, and subjected to proteomic analysis. Intriguingly, many C. zofingiensis LD proteins had no orthologs present in LD proteome of the model alga Chlamydomonas reinhardtii. Seven novel LD proteins (i.e., two functionally unknown proteins, two caleosins, two lipases, and one l-gulonolactone oxidase) and the major LD protein (MLDP), which were all transcriptionally up-regulated by ND, were selected for further investigation. Heterologous expression in yeast demonstrated that all tested LD proteins were localized to LDs and all except the two functionally unknown proteins enabled yeast to produce more TAG. MLDP could restore the phenotype of mldp mutant strain and enhance TAG synthesis in wild-type strain of C. reinhardtii. Although MLDP and caleosins had a comparable abundance in LDs, they responded distinctly to ND at the transcriptional level. The two lipases, instead of functioning as TAG lipases, likely recycled polar lipids to support TAG synthesis. For the first time, we reported that l-gulonolactone oxidase was abundant in LDs and facilitated TAG accumulation. Moreover, we also proposed a novel working model for C. zofingiensis LDs. Taken together, our work unravels the unique characteristics of C. zofingiensis LDs and provides insights into algal LD biogenesis and TAG synthesis, which would facilitate genetic engineering of this alga for TAG improvement.