Recent progress in high-throughput droplet screening and sorting for bioanalysis.

Owing to its ability to isolate single cells and perform high-throughput sorting, droplet sorting has been widely applied in several research fields. Compared with flow cytometry, droplet allows the encapsulation of single cells for cell secretion or lysate analysis. With the rapid development of this technology in the past decade, various droplet sorting devices with high throughput and accuracy have been developed. A droplet sorter with the highest sorting throughput of 30,000 droplets per second was developed in 2015. Since then, increased attention has been paid to expanding the possibilities of droplet sorting technology and strengthening its advantages over flow cytometry. This review aimed to summarize the recent progress in droplet sorting technology from the perspectives of device design, detection signal, actuating force, and applications. Technical details for improving droplet sorting through various approaches are introduced and discussed. Finally, we discuss the current limitations of droplet sorting for single-cell studies along with the existing gap between the laboratory and industry and provide our insights for future development of droplet sorters.

Modular remodeling of sterol metabolism for overproduction of 7-dehydrocholesterol in engineered yeast.

Vitamin D3 is a fat-soluble vitamin essential for the human body, and the biosynthesis of its precursor, 7-dehydrocholesterol (7-DHC), gains extensive attention. In this work, six genes (tHMG1, IDI1, ERG1, ERG11, ADH2, ERG7) and a transcription factor mutant UPC2G888A were overexpressed, increasing the 7-DHC titer from 1.2 to 115.3 mg/L. The CRISPR-mediated activation and repression systems were constructed and applied to the synthesis of 7-DHC, increasing the 7-DHC titer to 312.4 mg/L. Next, enzymes were compartmentalized into the endoplasmic reticulum (ER) and the ER lumen was enlarged by overexpressing INO2. The 7-DHC titer of the finally engineered yeast reached 455.6 mg/L in a shake flask and 2870 mg/L in a 5 L bioreactor, the highest 7-DHC titer reported so far. Overall, this study achieved a highly efficient 7-DHC synthesis by remodeling the complicated sterol synthesis modules, paving the way for large-scale 7-DHC bioproduction in the future.

Engineered yeast for efficient de novo synthesis of 7-dehydrocholesterol.

The synthesis of vitamin D3 precursor 7-dehydrocholesterol (7-DHC) by microbial fermentation has much attracted attention owing to its advantages of environmental protection. In this study, Saccharomyces cerevisiae was engineered for a de novo biosynthesis of 7-DHC. First, seven essential genes (six endogenous genes and one heterologous gene) were overexpressed, and the ROX1 gene (heme-dependent repressor of hypoxic genes) was knocked out. The resulting strain produced 82.6 mg/L 7-DHC from glucose. Then, we predicted five gene knockout targets for 7-DHC overproduction by the reconstruction of genome-scale metabolic model. GDH1 gene knockout increased the 7-DHC titer from 82.6 to 101.5 mg/L, and the specific growth rate of the ΔGDH1 mutant was also increased by 28%. Next, Ty1 transposon in S. cerevisiae was applied to increase the copies of the ERG1 gene and DHCR24 gene, resulting in a 120% increase in 7-DHC titer to 223.3 mg/L. Besides, to optimize the metabolic flux distribution, Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system was used to dynamically inhibit the competitive pathway, and the best binding site of ERG6 (delta (24)-sterol C-methyltransferase) promoter was screened out. The OD600 value of ERG6 regulated cells increased by 43% than knocking out ERG6 directly, and 7-DHC titer increased to 365.5 mg/L in a shake flask. Finally, the 7-DHC titer reached 1328 mg/L in 3-L bioreactor and the specific titer of 7-DHC reached up to 114.7 mg/g dry cell weight). Overall, this study constructed a yeast chassis for the highly efficient production of 7-DHC by systems metabolic engineering.

Sodium Arsenite-Induced Learning and Memory Impairment Is Associated with Endoplasmic Reticulum Stress-Mediated Apoptosis in Rat Hippocampus.

Chronic arsenic exposure has been associated to cognitive deficits. However, mechanisms remain unknown. The present study investigated the neurotoxic effects of sodium arsenite in drinking water over different dosages and time periods. Based on results from the Morris water maze (MWM) and morphological analysis, an exposure to sodium arsenite could induce neuronal damage in the hippocampus, reduce learning ability, and accelerate memory impairment. Sodium arsenite significantly increased homocysteine levels in serum and brain. Moreover, sodium arsenite triggered unfolded protein response (UPR), leading to the phosphorylation of RNA-regulated protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2 subunit α (eIF2α), and the induction of activating transcription factor 4 (ATF4). Arsenite exposure also stimulated the expression of the endoplasmic reticulum (ER) stress markers, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and the cleavage of caspase-12. Furthermore, exposure to arsenite enhanced apoptosis as demonstrated by expression of caspase-3 and TUNEL assay in the hippocampus. The results suggest that exposure to arsenite can significantly decrease learning ability and accelerate memory impairment. Potential mechanisms are related to enhancement of homocysteine and ER stress-induced apoptosis in the hippocampus.

Role of PTEN-Akt-CREB Signaling Pathway in Nervous System impairment of Rats with Chronic Arsenite Exposure.

The nervous system is a target of arsenic toxicity. Phosphatase and tensin homologue deleted on chromosome 10/protein kinase B/cAMP-response element binding protein (PTEN/Akt/CREB) signaling pathway has been reported to be involved in maintaining normal function of the nervous system, modulating growth and proliferation of neurocyte, regulating neuron synaptic plasticity, and long-term memory. And many studies have demonstrated that expressions of PTEN, Akt, and CREB protein were influenced by arsenic, but it is not clear whether this signaling pathway is involved in the nervous system impairment of rats induced by chronic arsenite exposure, and we have addressed this in this study. Eighty male Sprague-Dawley (SD) rats were randomly divided into eight groups (n = 10 each), four groups exposed to NaAsO2 (0, 5, 10, and 50 mg/L NaAsO2 in drinking water) for 3 months, the other four groups exposed to NaAsO2 (0, 5, 10, 50 mg/L NaAsO2 in drinking water) for 6 months. Hematoxylin and eosin (HE) staining showed that chronic arsenite exposure induced varying degrees of damage in cerebral neurons. And arsenite exposure increased arsenic amount in serum and brain samples in a dose- and time-dependent manner. Moreover, the protein levels of PTEN and Akt in brain tissue were not significantly changed compared with the control group, but p-Akt, CREB, and p-CREB were all significantly downregulated in arsenite-exposed groups with a dose-dependent pattern. These results suggested that chronic arsenite exposure negatively regulated the PTEN-Akt-CREB signaling pathway, and dysfunction of the signaling pathway might be one of the mechanisms of nervous system impairment induced by chronic arsenite exposure.