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.

Shape-memory microfluidic chips for fluid and droplet manipulation.

Fluid manipulation is an important foundation of microfluidic technology. Various methods and devices have been developed for fluid control, such as electrowetting-on-dielectric-based digital microfluidic platforms, microfluidic pumps, and pneumatic valves. These devices enable precise manipulation of small volumes of fluids. However, their complexity and high cost limit the commercialization and widespread adoption of microfluidic technology. Shape memory polymers as smart materials can adjust their shape in response to external stimuli. By integrating shape memory polymers into microfluidic chips, new possibilities for expanding the application areas of microfluidic technology emerge. These shape memory polymers can serve as actuators or regulators to drive or control fluid flow in microfluidic systems, offering innovative approaches for fluid manipulation. Due to their unique properties, shape memory polymers provide a new solution for the construction of intelligent and automated microfluidic systems. Shape memory microfluidic chips are expected to be one of the future directions in the development of microfluidic technology. This article offers a summary of recent research achievements in the field of shape memory microfluidic chips for fluid and droplet manipulation and provides insights into the future development direction of shape memory microfluidic devices.

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.

Vertical variation of bacterial production and potential role in oxygen loss in the southern Bay of Bengal.

Marine environments wherein long-term microbial oxygen consumption exceeds oxygen replenishment can be associated with oxygen minimum zones (OMZ). The Bay of Bengal OMZ (BOB-OMZ) is one of the most intense OMZs globally. To assess the contribution of bacterial oxygen consumption to oxygen loss in BOB-OMZ, we measured bacterial production (BP), temperature, salinity, and dissolved oxygen (DO) in the whole water column. We then compared the estimated bacterial oxygen demand (BOD) with diapycnal oxygen supply (DOS) at depths of 50-200 m in the southern BOB in January 2020. The average BP was 3.53 ± 3.15 µmol C m-3 h-1 in the upper 200 m of four stations, which was lower than those reported in other tropical waters. The vertical distribution of BP differed between the open ocean and nearshore areas. In the open ocean, temperature and DO were the most important predictors for BP in the whole water column. In the nearshore areas, when DO increased sharply from the suboxic state, extremely high BP occurred at 200 m. The average estimated BOD/DOS could reach up to 153% at depths of 50-200 m, indicating advection and anticyclonic eddies probably are important DO replenishment pathways in the BOB.

Characterization and anticancer effects of extracellular polysaccharide from DHA-producing microalga Crypthecodinium sp. SUN.

The Characterization and anticancer effects of extracellular polysaccharide (EPS) from DHA-producing microalga Crypthecodinium sp. SUN were studied in the present research. Results showed that EPS from C. sp. SUN have a molecular weight of 1.118 × 106 g/mol. EPS significantly inhibited the proliferation and migration of LA795 lung adenocarcinoma cells, and the apoptosis rate decreased in a concentration-dependent manner, reached 52 % at 15 mg/mL. C. sp. SUN EPS also significantly decreased reactive oxygen species (ROS) level by over 50 %, superoxide dismutase (SOD) activity by 76 %, and catalase (CAT) activity by 34 % at 10 mg/mL, indicating that EPS may inhibit tumor cell growth instead of killing tumor cells. Additionally, C. sp. SUN EPS suppressed cell proliferation by downregulating the expression of adhesion proteins and cyclin D1 in LA795 cells. In vivo experiments demonstrated that C. sp. SUN EPS inhibited the growth of lung adenocarcinoma tumors without affecting the normal body weight of nude mice. Collectively, the present study showed that C. sp. SUN EPS could be a potential substance for cancer treatment, which provided a research basis for future study on EPS and expanded the application of Crypthecodinium.

Open-channel microfluidic chip based on shape memory polymer for controllable liquid transport.

Open microfluidics has attracted increasing attention over the last decade because of its flexibility and simplicity with respect to cell culture and clinical diagnosis. However, traditional valves and pumps are difficult to integrate on open-channel microfluidic chips, in which a liquid is usually driven by capillary forces. Poor fluid control performance is a common drawback of open microfluidics. Herein, we proposed a method for controlling the liquid flow in open channels by controlling the continuous Laplace pressure induced by the deformation of the shape memory microstructures. The uniformly arranged cuboidal microcolumns in the open channels have magnetic/light dual responses, and the bending angle of the microcolumns can be controlled by adjusting Laplace pressure using near-infrared laser irradiation in a magnetic field. Laplace pressure and capillary force drove the liquid flow together, and the controllable fluid transport was realized by adjusting the hydrophilicity of the channel surface and the bending angle of the microcolumns. We demonstrated the controllability of the flow rate and the directional transport of water along a preset path. In addition, the start and stop of water transport were realized via local hydrophobic modification. The proposed strategy improves poor fluid control in traditional open systems and makes fluid flow highly controllable. We tried to extract and detect rhodamine B in tiny droplets on the open microfluidic chip, demonstrating the advantages of the proposed strategy in the separation and analysis of tiny samples.

Photothermal visual sensing of alkaline phosphatase based on the etching of Au@MnO2 core-shell nanoparticles.

Alkaline phosphatase (ALP), as a crucial enzyme involved in many physiological activities, is always used as one of the significant biomarkers in clinical diagnosis. Herein, a novel, simple, and effective photothermal quantitative method based on the etching of MnO2-coated gold nanoparticles (Au@MnO2 NPs) was established for ALP activity assay with a household thermometer-based visual readout. The photothermal effect of Au@MnO2 NPs is much higher than that of MnO2 NPs or Au NPs. The MnO2 shell of Au@MnO2 NPs can be etched by ascorbic acid, a product of ALP-catalyzed hydrolysis of 2-phospho-l-ascorbic acid. With the etching of Au@MnO2 NPs, the photothermal conversion efficiency decreased gradually, causing the decrease of the temperature increment of the solutions by degrees. A household thermometer, instead of large-scale and professional instruments, was used as a signal reader to realize the visual quantitative detection. The photothermal platform was used successfully for the determination of ALP with a wide linear range from 2.0 to 50 U/L and a detection limit as low as 0.75 U/L. Moreover, the inhibition efficiency of sodium vanadate for ALP activity was investigated, proving the photothermal quantitative method will be a potential platform for screening enzyme inhibitors. Such a sensitive, facile, and low-cost sensing assay provides a new prospect to develop platforms for point-of-care testing.

Tracking cellular transformation of As(III) in HepG2 cells by single-cell focusing/capillary electrophoresis coupled to ICP-MS.

The cellular metabolism of metals is highly critical to elucidate their potential cytotoxicity or cell protection mechanism. In this work, an asymmetric serpentine microfluidic device (ASMD) with high sampling efficiency and excellent focusing performance was developed for single-cell focusing. ASMD coupling with ICP-MS ensures single-cell assay to provide the information for trivalent arsenic (As(III)) uptake by HepG2 cells, which reveals the heterogeneity of cellular arsenic distribution, and elucidates the arsenic elimination behaviors in single HepG2 cells. Further, the metabolism and transformation of As(III) in HepG2 cells was tracked by hyphenating capillary electrophoresis (CE) separation with ICP-MS. The results for single-cell analysis and arsenic elimination kinetics illustrated that the half-life of arsenic elimination is 0.9 ± 0.04 h with the elimination constant of 0.77 ± 0.03, i.e., 77% of accumulated As in HepG2 cells may be eliminated per hour. Moreover, arsenobetaine (AsB) was identified to be the main metabolite and biotransformation species of As in HepG2 cells. ASMD-ICP-MS and CE-ICP-MS are powerful for tracking the fate of metals or metal drugs in single cells to comprehensively understand their metabolic pathway and transformation behaviors.

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.

In situ monitoring PUVA therapy by using a cell-array chip-based SERS platform.

Psoralen ultraviolet A (PUVA) therapy has thrived as a promising treatment for psoriasis. However, overdose of PUVA treatment will cause side-effects, such as melanoma formation. And these side-effects are often ignored during PUVA therapy. Hence, in situ monitoring therapeutic response of PUVA therapy is important to minimize side-effects. Aberrant expression of tyrosinase (TYR) has been proved to be associated with melanoma, indicating that TYR is a potential target for evaluation of PUVA therapy. Herein, we reported a strategy for in situ monitoring TYR activity during PUVA therapy by using a cell-array chip-based SERS platform. The cell-array chip was used to simulate cell survival environment for cell culture. Capture of single cells and living cell analysis were realized in the isolated microchambers. An enzyme-induced core-shell self-assembly substrate was used to evaluate TYR activity in living cells during PUVA therapy. The gold nanoparticle modified with a SERS reporter, 4-mercaptobenzonitrile (4-MBN), was used as the core. In the presence of oxygen and TYR, hydroxylation of l-tyrosine occurred, leading to the reduction of silver ion on the surface of gold cores. The growth of silver shells was accompanied by the increased SERS intensity of the reporter, which is related directly to TYR activity. The detection limit for TYR activity is 0.45 U/mL. Upregulation of TYR activity was successfully monitored after PUVA therapy. Notably, real-time and in situ information of therapeutic response can be obtained through monitoring PUVA therapy by using a cell-array chip-based SERS platform, which has great potential to guide the clinical application of PUVA therapy.

A digital microfluidic platform based on a near-infrared light-responsive shape-memory micropillar array.

In this work, we developed a digital microfluidic platform based on a shape memory micropillar array responsive to near-infrared light, and the droplets were programmatically manipulated through light-induced micropillar deformation. The micropillar array was constructed on the surface of a poly(ethylene-vinyl acetate) copolymer, a shape memory polymer sensitive to near-infrared light. Before droplet manipulation, the micropillar array was kept temporarily tilted by heating and pressing. Under the irradiation of a near-infrared laser, the micropillar array achieved the transition from the temporary shape to the original shape. Temperature gradient and micropillar deformation caused by near-infrared light irradiation produce the driving force for droplet movement. The movement of the laser mounted on an electronically controlled displacement platform was controlled by a computer to achieve the programmed control of the droplets. Moreover, we demonstrated light-manipulated droplet movement and fusion, and achieved ascorbic acid detection using this digital microfluidic platform. In particular, the micropillar array chip is able to manipulate droplets in a wide range of 0.1 µL to 10 µL. The proposed digital microfluidic platform will broaden the application of digital microfluidic technology in analytical chemistry and biomedicine.

Inertial-Force-Assisted, High-Throughput, Droplet-Free, Single-Cell Sampling Coupled with ICP-MS for Real-Time Cell Analysis.

Single-cell analysis facilitates perception into the most essential processes in life's mysteries. While it is highly challenging to quantify them at the single-cell level, where precise single-cell sampling is the prerequisite. Herein, a real-time single-cell quantitative platform was established for high-throughput droplet-free single-cell sampling into time-resolved (TRA) ICP-MS and real-time quantification of intracellular target elements. The concentrated cells (2 × 106 cells mL-1) were spontaneously and orderly aligned in a spiral microchannel with 104 periodic dimensional confined micropillars. The quantification is conducted simultaneously by internal standard inducing from another branch channel in the chip. The flow-rate-independent feature of single-cell focusing into an aligned stream within a wide range of fluidic velocities (100-800 µL min-1) facilitates high-throughput, oil-free, single-cell introduction into TRA-ICP-MS. The system was used for real-time exploration of intracellular antagonism of Cu2+ against Cd2+. an obvious antagonistic effect was observed for the MCF-7 cell by culturing for 3, 6, 9, and 12 h with 100 µg L-1 Cd2+ and 100 µg L-1 Cu2+, and a rivalry rate of 12.8% was achieved at 12 h. At identical experimental conditions, however, limited antagonistic effect was encountered for a bEnd3 cell within the same incubation time period, with a rivalry rate of 4.81%. On the contrary, an antagonistic effect was not observed for the HepG2 cell by culturing for 6 h, while an obvious antagonistic effect was found by further culturing to 12 h, with a rivalry rate of 10.43%. For all three cell lines, significant heterogeneity was observed among individual cells.

Multiplexed detection of micro-RNAs based on microfluidic multi-color fluorescence droplets.

In this work, simple, rapid, and low-cost multiplexed detection of tumor-related micro-RNAs (miRNAs) was achieved based on multi-color fluorescence on a microfluidic droplet chip, which simplified the complexity of light path to a half. A four-T-junction structure was fabricated to form uniform nano-volume droplet arrays with customized contents. Multi-color quantum dots (QDs) used as the fluorescence labels were encapsulated into droplets to develop the multi-path fluorescence detection module. We designed an integrated multiplex fluorescence resonance energy transfer system assisted by multiple QDs (four colors) and one quencher to detect four tumor-related miRNAs (miRNA-20a, miRNA-21, miRNA-155, and miRNA-221). The qualitative analysis of miRNAs was realized by the color identification of QDs, while the quantitative detection of miRNAs was achieved based on the linear relationship between the quenching efficiency of QDs and the concentration of miRNAs. The practicability of the multiplex detection device was further confirmed by detecting four tumor-related miRNAs in real human serum samples. The detection limits of four miRNAs ranged from 35 to 39 pmol/L was achieved without any target amplification. And the linear range was from 0.1 nmol/L to 1 µmol/L using 10 nL detection volume (one droplet) under the detection speed of 320 droplets per minute. The multiple detection system for miRNAs is simple, fast, and low-cost and will be a powerful platform for clinical diagnostic analysis. Graphical abstract.

Characterization of cysteine sulfinic acid decarboxylase from Tribolium castaneum and its application in the production of ß-alanine.

ß-alanine is a precursor for the production of pharmaceuticals and food additives that is produced by chemical methods in industry. As concerns about the environment and energy are increasing, biocatalysis using L-aspartate-α-decarboxylase (ADC) to convert L-aspartate to ß-alanine has great potential. Many studies have focused on the catalytic activity of ADC, but these researches were limited to the prokaryotic enzymes. In this study, the gene encoding cysteine sulfinic acid decarboxylase from Tribolium castaneum (TcCSADC) was synthesized and overexpressed in Escherichia coli, and the enzyme was purified and characterized for the first time. It could use L-aspartate as its substrate, and the specific activity was 4.83 µmol/min/mg, which was much higher than that of ADCs from prokaryotes. A homology modeling assay indicated that TcCSADC had a dimer structure. Based on the evolutionary information from thermophilic bacteria, twenty-three variants were constructed to attempt to improve its abilities that transform L-aspartate to ß-alanine. One mutant, G369A, was screened that had improved thermal stability. An analysis of the suitability of the catalytic process showed that the up to 162 g/L ß-alanine could be produced using cells expressing the recombinant G369A variant, which is the highest yield to date. The CSADC from T. castaneum has important value for studies of the mechanism of ADCs and CSADCs from eukaryotes, and the engineered strain containing the G369A variant has great potential for the industrial production of ß-alanine.

SERS monitoring of the Fenton degradation reaction based on microfluidic droplets and alginate microparticles.

Surface-enhanced Raman scattering (SERS) spectroscopy as a powerful tool has been used to explore different catalysis degradation reactions, whereas some drawbacks caused by ferric ions still exist in the current SERS monitoring of the Fenton reaction process. In this work, microfluidic droplet- and alginate microparticle-based methods were, respectively, applied to realize SERS monitoring of the Fenton degradation process in a relatively stable environment, which benefited from reduction of the loss of ferrous ions and the aggregation of the SERS substrate. As expected, the spectroscopic evidence at the molecular level directly revealed the degradation mechanism of rhodamine dyes, showing that the chemical bonds between xanthene and carboxybenzene broke continuously during the reaction. Afterward, the degradation mechanism determined by SERS was verified via mass spectrometry detection, which confirmed the validity of the SERS-based method. More broadly, the microfluidic droplet- and microparticle-based methods are potentially applicable for SERS monitoring of more Fenton degradation reactions.

Simultaneous quantitative detection of multiple tumor markers in microfluidic nanoliter-volume droplets.

We developed a simultaneous detection method for multiple tumor markers (TMs) in microfluidic droplets based on a multiple fluorescence resonance energy transfer (FRET) system. In this system, graphene oxide (GO) was used as the single quencher, while the multi-color quantum dots (QDs) labeled on different aptamers were employed as energy donors. When the aptamers were adsorbed onto GO due to the π-π stacking interaction, QDs were drawn to the surface of GO and quenched by it. Once the TMs were introduced, the corresponding fluorescence of QDs was recovered obviously owing to the preferential interaction of aptamers with the TMs. Here, the multi-FRET system was encapsulated into nanoliter-volume droplets by a simple T-junction microfluidic chip. The targets could be detected rapidly as the generated droplets flew through the integrated on-line detection zone. Three tumor markers, carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and vascular endothelial growth factor (VEGF165) could be detected simultaneously in 33 nL-volume droplets, which is only 1/3000 of the volume of the sample consumed in the conventional fluorescence spectrophotometer. In addition, the signals corresponding to different TM targets in one nanoliter-volume droplet could be read out at the same time, and the signals could be output continuously owing to the uninterruptible generation of droplets. Even with a signal acquisition frequency of 55 droplets per minute, the multi-FRET biosensing system has linear ranges of 0.50-70 ng mL-1 for CEA, 0.25-70 ng mL-1 for PSA and 0.50-70 ng mL-1 for VEGF165. The detection limits of CEA, PSA and VEGF165 were calculated to be 0.15 ng mL-1, 0.035 ng mL-1 and 0.11 ng mL-1, respectively. The method was also validated by analyzing human serum sample dilutions. The proposed multi-FRET-based system has potential to become a powerful tool for rapid, low-cost and simultaneous detection of multiple tumor markers.