Reactive oxygen species derived from NADPH oxidase as signaling molecules regulate fatty acids and astaxanthin accumulation in Chromochloris zofingiensis.

Abiotic stresses can increase the total fatty acid (TFA) and astaxanthin accumulation in microalgae. However, it remains unknown whether a unified signal transduction mechanism exists under different stresses. This study explored the link between nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) and the accumulation of fatty acids and astaxanthin in Chromochloris zofingiensis under three abiotic stresses. Results showed significant increases in fatty acid, astaxanthin, and ROS levels under nitrogen deficiency, phosphorus deficiency, and high-salinity stress. The introduction of the NADPH oxidase inhibitor diphenyleneiodonium (DPI) decreased the content of these components. This underscores the pivotal role of NADPH oxidase-derived ROS in the accumulation of fatty acid and astaxanthin under abiotic stress. Analysis of transcriptomes across three conditions following DPI addition revealed 1,445 shared differentially expressed genes (DEGs). Enrichment analysis revealed that biotin, betalain, thiamine, and glucosinolate may be important in stress responses. The heatmap demonstrated that DPI notably suppressed gene expression in the fatty acid and carotenoid biosynthesis pathways. Our findings underscore the pivotal role of NADPH oxidase-derived ROS in the accumulation of fatty acid and astaxanthin under abiotic stresses.

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.

The metabolic mechanisms of Cd-induced hormesis in photosynthetic microalgae, Chromochloris zofingiensis.

Cadmium, an environmental pollutant, is highly toxic and resistant to degradation. It exhibits toxicity at elevated doses but triggers excitatory effects at low doses, a phenomenon referred to as hormesis. Microalgae, as primary producers in aquatic ecosystems, demonstrate hormesis induced by cadmium, though the specific mechanisms are not yet fully understood. Consequently, we examined the hormesis of cadmium in Chromochloris zofingiensis. A minimal Cd2+ concentration (0.05 mg L-1) prompted cell proliferation, whereas higher concentrations (2.50 mg L-1) inhibited growth. The group exposed to higher doses exhibited increased levels of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT). Contrastingly, the group exposed to low doses exhibited a moderate antioxidant response without significantly increasing ROS. This implies that increased levels of antioxidative components counteract excessive ROS, maintaining cellular redox balance and promoting growth under conditions of low Cd2+. Validation experiments have established that NADPH oxidase-derived ROS primarily coordinates the hormesis effect in microalgae. Comparative transcriptome analysis has proved the involvement of antioxidant systems and photosynthesis in regulating hormesis. Notably, Aurora A kinases consistently displayed varying expression levels across all Cd2+ treatments, and their role in microalgal hormesis was confirmed through validation with SNS-314 mesylate. This study unveils the intricate regulatory mechanisms of Cd-induced hormesis in C. zofingiensis, with implications for environmental remediation and industrial microalgae applications.

Imaging microglia surveillance during sleep-wake cycles in freely behaving mice.

Microglia surveillance manifests itself as dynamic changes in cell morphology and functional remodeling. Whether and how microglia surveillance is coupled to brain state switches during natural sleep-wake cycles remains unclear. To address this question, we used miniature two-photon microscopy (mTPM) to acquire time-lapse high-resolution microglia images of the somatosensory cortex, along with EEG/EMG recordings and behavioral video, in freely-behaving mice. We uncovered fast and robust brain state-dependent changes in microglia surveillance, occurring in parallel with sleep dynamics and early-onset phagocytic microglial contraction during sleep deprivation stress. We also detected local norepinephrine fluctuation occurring in a sleep state-dependent manner. We showed that the locus coeruleus-norepinephrine system, which is crucial to sleep homeostasis, is required for both sleep state-dependent and stress-induced microglial responses and ß2-adrenergic receptor signaling plays a significant role in this process. These results provide direct evidence that microglial surveillance is exquisitely tuned to signals and stressors that regulate sleep dynamics and homeostasis so as to adjust its varied roles to complement those of neurons in the brain. In vivo imaging with mTPM in freely behaving animals, as demonstrated here, opens a new avenue for future investigation of microglia dynamics and sleep biology in freely behaving animals.

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.

Encoding of social novelty by sparse GABAergic neural ensembles in the prelimbic cortex.

Although the prelimbic (PrL) area is associated with social behaviors, the neural ensembles that regulate social preference toward novelty or familiarity remain unknown. Using miniature two-photon microscopy (mTPM) to visualize social behavior-associated neuronal activity within the PrL in freely behaving mice, we found that the Ca2+ transients of GABAergic neurons were more highly correlated with social behaviors than those of glutamatergic neurons. Chemogenetic suppression of social behavior-activated GABAergic neurons in the PrL disrupts social novelty behaviors. Restoring the MeCP2 level in PrL GABAergic neurons in MECP2 transgenic (MECP2-TG) mice rescues the social novelty deficits. Moreover, we identified and characterized sparsely distributed NewPNs and OldPNs of GABAergic interneurons in the PrL preferentially responsible for new and old mouse exploration, respectively. Together, we propose that social novelty information may be encoded by the responses of NewPNs and OldPNs in the PrL area, possibly via synergistic actions on both sides of the seesaw.

Simultaneous vessel segmentation and unenhanced prediction using self-supervised dual-task learning in 3D CTA (SVSUP).

BACKGROUND AND OBJECTIVE: The vessel segmentation in CT angiography (CTA) provides an important basis for automatic diagnosis and hemodynamics analysis. Virtual unenhanced (VU) CT images obtained by dual-energy CT can assist clinical diagnosis and reduce radiation dose by obviating true unenhanced imaging (UECT). However, accurate segmentation of all vessels in the head-neck CTA (HNCTA) remains a challenge, and VU images are currently not available from conventional single-energy CT imaging. METHODS: In this paper, we proposed a self-supervised dual-task deep learning strategy to fully automatically segment all vessels and predict unenhanced CT images from single-energy HNCTA based on a developed iterative residual-sharing scheme. The underlying idea was to use the correlation between the two tasks to improve task performance while avoiding manual annotation for model training. RESULTS: The feasibility of the strategy was verified using the data of 24 patients. For vessel segmentation task, the proposed model achieves a significantly higher average Dice coefficient (84.83%, P-values 10-3 in paired t-test) than the state-of-the-art segmentation model, vanilla VNet (78.94%), and several popular 3D vessel segmentation models, including Hessian-matrix based filter (62.59%), optically-oriented flux (66.33%), spherical flux model (66.91%), and deep vessel net (66.47%). For the unenhanced prediction task, the average ROI-based error compared to the UECT in the artery tissue is 6.1±4.5 HU, similar to previously reported 6.4±5.1 HU for VU reconstruction. CONCLUSIONS: Results show that the proposed dual-task framework can effectively improve the accuracy of vessel segmentation in HNCTA, and it is feasible to predict the unenhanced image from single-energy CTA, providing a potential new approach for radiation dose saving. Moreover, to our best knowledge, this is the first reported annotation-free deep learning-based full-image vessel segmentation for HNCTA.

Dynamic R2' Imaging can Be a Biomarker for Diagnosing and Staging Early Acute Kidney Injury in Animals.

Background: Early diagnosis of acute kidney injury (AKI) is essential in clinical settings. None of the current biomarkers are widely applied. The combination of pulse-shifting multi-echo asymmetric spin-echo sequence (psMASE) and a modified hemodynamic response imaging (HRI) technique is promising. The purpose of this study was to evaluate the feasibility of psMASE combined with HRI in detecting early ischemic AKI in animal models of different severities. Methods: Twenty rabbits were divided into four groups (mild, moderate, and severe AKI and control groups). Transarterial embolization with different doses of microspheres was performed to establish AKI animal models of different severities. The 3T psMASE and HRI scans of kidneys were conducted. The R2*, R2, and R2' during room air and gas stimulation were acquired and the difference of R2' (dR2') was evaluated in different AKI groups. Results: The values were not different in R2* and R2 during room air and in R2* and R2, and R2' during gas stimulation. The value of R2' was significantly different during room air (P = 0.014), but the difference was only found between control and moderate/severe AKI groups (P = 0.032 and 0.022). The values of dR2' were different among groups (P < 0.0001) and differences between every two groups except comparison of moderate and severe AKI groups were significant (P < 0.01). Conclusion: The dR2' imaging acquired by a combination of renal psMASE and HRI technique can serve as a potential quantitative biomarker for early detection and staging of AKI.

Light-sheet fluorescence imaging charts the gastrula origin of vascular endothelial cells in early zebrafish embryos.

It remains challenging to construct a complete cell lineage map of the origin of vascular endothelial cells in any vertebrate embryo. Here, we report the application of in toto light-sheet fluorescence imaging of embryos to trace the origin of vascular endothelial cells (ECs) at single-cell resolution in zebrafish. We first adapted a previously reported method to embryo mounting and light-sheet imaging, created an alignment, fusion, and extraction all-in-one software (AFEIO) for processing big data, and performed quantitative analysis of cell lineage relationships using commercially available Imaris software. Our data revealed that vascular ECs originated from broad regions of the gastrula along the dorsal-ventral and anterior-posterior axes, of which the dorsal-anterior cells contributed to cerebral ECs, the dorsal-lateral cells to anterior trunk ECs, and the ventral-lateral cells to posterior trunk and tail ECs. Therefore, this work, to our knowledge, charts the first comprehensive map of the gastrula origin of vascular ECs in zebrafish, and has potential applications for studying the origin of any embryonic organs in zebrafish and other model organisms.