Regulatory mechanisms of autophagy on DHA and carotenoid accumulation in Crypthecodinium sp. SUN.

BACKGROUND: Autophagy is a crucial process of cellular self-destruction and component reutilization that can affect the accumulation of total fatty acids (TFAs) and carotenoids in microalgae. The regulatory effects of autophagy process in a docosahexaenoic acid (DHA) and carotenoids simultaneously producing microalga, Crypthecodinium sp. SUN, has not been studied. Thus, the autophagy inhibitor (3-methyladenine (MA)) and activator (rapamycin) were used to regulate autophagy in Crypthecodinium sp. SUN. RESULTS: The inhibition of autophagy by 3-MA was verified by transmission electron microscopy, with fewer autophagy vacuoles observed. Besides, 3-MA reduced the glucose absorption and intracellular acetyl-CoA level, which resulting in the decrease of TFA and DHA levels by 15.83 and 26.73% respectively; Surprisingly, 3-MA increased intracellular reactive oxygen species level but decreased the carotenoids level. Comparative transcriptome analysis showed that the downregulation of the glycolysis, pentose phosphate pathway and tricarboxylic acid cycle may underlie the decrease of acetyl-CoA, NADPH and ATP supply for fatty acid biosynthesis; the downregulation of PSY and HMGCR may underlie the decreased carotenoids level. In addition, the class I PI3K-AKT signaling pathway may be crucial for the regulation of carbon and energy metabolism. At last, rapamycin was used to activate autophagy, which significantly enhanced the cell growth and TFA level and eventually resulted in 1.70-fold increase in DHA content. CONCLUSIONS: Our findings indicate the mechanisms of autophagy in Crypthecodinium sp. SUN and highlight a way to manipulate cell metabolism by regulating autophagy. Overall, this study provides valuable insights to guide further research on autophagy-regulated TFA and carotenoids accumulation in Crypthecodinium sp. SUN.

Low and high temperatures promote docosahexaenoic acid accumulation in Crypthecodinium sp. SUN by regulating the polyunsaturated fatty acid synthase pathway and the expression of saturated fatty acid preferred diacylglycerol acyltransferases.

Low (15 °C) and high (35 °C) temperatures significantly increased DHA as a percentage of total fatty acids (TFAs) to 43.6 % and 40.46 %, respectively (1.28- and 1.18-fold of that at 25 °C, respectively). The incompleteness of the FAS pathway indicates that DHA synthesis does not occur via this pathway. Meanwhile, Comparative transcriptome analysis showed that the PUFA synthase pathway might be responsible for DHA synthesis in C. sp. SUN. Additionally, the three diacylglycerol acyltransferases all had a substrate preference for saturated fatty acid (SFA)-CoA, which also contributed to the decreased SFA and increased DHA at both low and high temperatures. Additionally, WGCNA analysis identifies key regulatory genes that may be involved in temperature-regulated DHA proportion. The findings of this study indicate the mechanisms of temperature-regulated DHA accumulation in C. sp. SUN and shed light on the manipulation of DHA proportion by changes in temperature.

Treatment of nitrogen and phosphorus in wastewater by heterotrophic N- and P-starved microalgal cell.

Nitrogen (N) and phosphorus (P) are two major pollutants present in aquaculture wastewater, and their concentrations often do not meet discharge standards. In the present study, the N and P removal efficiency of nutrient-deficient cells (S group) was significantly higher than that of photoautotrophic cells (P group) and heterotrophic cells (H group). After incubation with wastewater, the N and P content of S group cells was significantly increased and reached a level similar to that of the P group and H group cells after 6 days of treatment. Additionally, in the S group cells, the content of total fatty acids (TFAs), which can be used to supply energy and organic carbon for N and P absorption, significantly decreased. In addition, the protein and nucleic acid contents of the S group cells also significantly increased, which revealed the biosynthetic flow of assimilated N and P. Comparative transcriptome analysis showed that compared with the P group and H group, the N metabolism, ribosome, RNA polymerase, and fatty acid degradation pathways were significantly upregulated in the S group cells, and the fatty acid biosynthesis pathway was significantly downregulated, which was in agreement with the biochemical results. In summary, our study showed that N- and P-starved heterotrophic cells are ideal for use in wastewater N and P removal processes. Keypoints • The N and P removal efficiencies of the S group were higher than P and H groups • Fatty acids were degraded to supply energy and carbon for N and P absorption • N metabolism and fatty acid degradation pathways were upregulated in the S group.

Mechanisms of Sodium-Acetate-Induced DHA Accumulation in a DHA-Producing Microalga, Crypthecodinium sp. SUN.

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (PUFA) that is critical for the intelligence and visual development of infants. Crypthecodinium is the first microalga approved by the Food and Drug Administration for DHA production, but its relatively high intracellular starch content restricts fatty acid accumulation. In this study, different carbon sources, including glucose (G), sodium acetate (S) and mixed carbon (M), were used to investigate the regulatory mechanisms of intracellular organic carbon distribution in Crypthecodinium sp. SUN. Results show that glucose favored cell growth and starch accumulation. Sodium acetate limited glucose utilization and starch accumulation but caused a significant increase in total fatty acid (TFA) accumulation and the DHA percentage. Thus, the DHA content in the S group was highest among three groups and reached a maximum (10.65% of DW) at 96 h that was 2.92-fold and 2.24-fold of that in the G and M groups, respectively. Comparative transcriptome analysis showed that rather than the expression of key genes in fatty acids biosynthesis, increased intracellular acetyl-CoA content appeared to be the key regulatory factor for TFA accumulation. Additionally, metabolome analysis showed that the accumulated DHA-rich metabolites of lipid biosynthesis might be the reason for the higher TFA content and DHA percentage of the S group. The present study provides valuable insights to guide further research in DHA production.

Effectiveness and safety of ultrasound-guided high-intensity focused ultrasound ablation for the treatment of colorectal cancer liver metastases.

OBJECTIVE: To investigate the effectiveness and safety of high-intensity focused ultrasound (HIFU) ablation for patients with colorectal liver metastases (CRLMs) who were unsuitable for hepatectomy. METHODS: This is a multicenter retrospective study. 238 CRLM patients underwent ultrasound-guided HIFU (USgHIFU) ablation in three medical centers from October 2014 to December 2020. Patients who had complete colorectal cancer resection, but exhibited extra-hepatic metastasis were excluded from this study. HIFU ablation procedure was performed, and contrast-enhanced MR imaging and/or contrast-enhanced CT examinations were conducted and mRECIST was used for the assessment of tumor ablation effectiveness before and after treatment, and every 3 months thereafter. Adverse events and complications were recorded. RESULTS: 43 CRML patients (27 male, 16 female, aged 29-82 years) were enrolled and underwent a USgHIFU ablation procedure. CR (complete response) was achieved in 21 patients, while PR (partial response) was observed in 21 patients and SD (stable disease) was achieved in one patient, respectively. The objective response rate was 97.7%. Median OS (overall survival) was estimated to be 31 months, and1-year and 18-month overall survival was 90.7% (39/43) and 72.1% (31/43), respectively. For CR and PR patients, the median OS was 35 months and 23 months, respectively (p = 0.00). The majority of adverse events were pain in 22 cases (51.2%) and local skin edema in 33 cases (76.7%). No severe adverse events or complications were reported and recorded. CONCLUSIONS: USgHIFU ablation is a safe and effective treatment option for CRLM patients, especially for patients who are unsuitable for hepatectomy.

Feasibility, Safety, and Efficacy of Accurate Uterine Fibroid Ablation Using Magnetic Resonance Imaging-Guided High-Intensity Focused Ultrasound With Shot Sonication.

The aim of this study was to investigate the feasibility, safety, and efficacy of uterine fibroid treatment using magnetic resonance imaging (MRI)-guided high-intensity focused ultrasound (US) with shot sonication for accurate ablation. Forty-three patients with 51 symptomatic uterine fibroids were treated with MRI-guided high-intensity focused US with shot sonication, which was a small acoustic focus of higher intensity with a shorter time (2 seconds) of US exposure and a shorter cooling time (2-3 seconds). The treatment efficacy and adverse events were analyzed, and the changes in the severity of symptoms and the reduction in fibroid volume were assessed 3 and 6 months after the procedure. All patients were successfully treated in a single session, without major complications, and the mean nonperfused volume ratio ± SD was 84.3% ± 15.7% (range, 33.8%-100%).Complete ablation was achieved in 13 T2-hypointense fibroids from 10 patients, and partial ablation was achieved in 38 fibroids from 33 patients. The overall mean treatment time was 135.0 ± 50.9 minutes (2.2 ± 0.8 hours). The transformed symptom severity scores and mean fibroid volumes decreased significantly after treatment (P < .05). In conclusion, MRI-guided high-intensity focused US with shot sonication is a feasible, safe, and effective technique for ablation of uterine fibroids and complete ablation of T2-hypointense fibroids.