Combining metabolomics and transcriptomics to analyze key response metabolites and molecular mechanisms of Aspergillus fumigatus under cadmium stress.

The co-cultivation of fungi with microalgae facilitates microalgae harvesting and enhances heavy metal adsorption. However, the mechanisms of fungal tolerance to cadmium (Cd) have not yet been studied in detail. In this study, functional groups of fungi were analyzed under Cd stress using Fourier transform infrared spectrometer (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM), and transmission electron microscope (TEM) to explore their morphology. Confocal laser scanning microscope (CLSM) was used to characterize the changes in the content of extracellular polysaccharides and proteins, and a decrease in the ratio of glutathione (GSH) to oxidized glutathione (GSSG) was monitored. The GSH and GSSG contents in mycelium were 7.4 and 7.9 times higher than that in the control, respectively. After 72 h of Cd treatment, the fungal extracellular polysaccharide and extracellular protein contents increased by 16 and 11.4 mg/g, respectively, compared to the control. This provided several functional groups for the complexation of Cd ions to enhance fungal Cd tolerance. The metabolomic and transcriptomic results revealed a total of 358 differential metabolites after 20, 48, and 72 h in the positive and negative ion modes, and the number of differential metabolites specific to each group was 104, 14, and 89, respectively. There were 927, 1167, and 1287 up-regulated genes, and 1301, 1480, and 1683 down-regulated genes at 20, 48, and 72 h, respectively. Energy metabolism, amino acid metabolism, and the ABC transport system are the key metabolic pathways for tolerance enhancement and heavy metal detoxification in fungi. The expression of S-cysteinosuccinic acid was significantly up-regulated after Cd stress and associated with enhanced fungal tolerance and resistance to Cd.

Physiological regulation of microalgae under cadmium stress and response mechanisms of time-series analysis using metabolomics.

The investigation of heavy metal wastewater treatment utilizing microalgae adsorption has been extensively demonstrated. However, the response mechanism based on metabolomics to analyze the time-series changes of microalgae under Cd stress has not been described in detail. In this study, SEM/TEM demonstrated that Cd accumulated on the cell surface of microalgae and was bioconcentrated in the cytoplasm, vesicles, and chloroplasts. Carbonyl/quinone/ketone/carboxyl groups (OCO), membrane polysaccharides (OH), and phospholipids (PO) were involved in the interaction of Cd ions, and the chlorophyll content underwent a process of decreasing in the early stage (1.62 mg/g at 48 h) and recovering to the normal level in the late stage, and the contents of MDA, GSH, and SOD were all increased (29.7 nmol/g, 0.23 mg/g, and 30.01 u/106 cells) and then gradually returned to the steady state. The results of EPS content and fluorescent labeling showed that Cd induced the overexpression and synthesis of extracellular polysaccharides and proteins, which is one of the defense mechanisms participating in the reduction of cellular damage by complexed Cd. Metabolomics results indicated that the malate synthesis pathway was activated after Cd-20 h, and the microalgal cells began to shift the metabolic pathway to storage lipid or polysaccharide biosynthesis. In the Calvin cycle, the expression of D-Sedoheptulose 7-phosphate in Cd-20 h_vs_ck and Cd-72 h_vs_Cd-20 h firstly declined and then increased, and the photosynthesis system was suppressed at the beginning, and then gradually returned to normal to maintain the successful development of the dark reaction. The results of time series analysis revealed that the response of microalgae to Cd was categorized into fast response and slow response to regulate cell adsorption and growth metabolism.

Real-time visualization of two-photon fluorescence lifetime imaging microscopy using a wavelength-tunable femtosecond pulsed laser.

A fluorescence lifetime imaging microscopy (FLIM) integrated with two-photon excitation technique was developed. A wavelength-tunable femtosecond pulsed laser with nominal pulse repetition rate of 76-MHz was used to acquire FLIM images with a high pixel rate of 3.91 MHz by processing the pulsed two-photon fluorescence signal. Analog mean-delay (AMD) method was adopted to accelerate the lifetime measurement process and to visualize lifetime map in real-time. As a result, rapid tomographic visualization of both structural and chemical properties of the tissues was possible with longer depth penetration and lower photo-damage compared to the conventional single-photon FLIM techniques.