High-precision screening and sorting of double emulsion droplets.

Mounting evidence suggests that cell populations are extremely heterogeneous, with individual cells fulfilling different roles within the population. Flow cytometry (FC) is a high-throughput tool for single-cell analysis that works at high optical resolution. Sub-populations with unique properties can be screened, isolated and sorted through fluorescence-activated cell sorting (FACS), using intracellular fluorescent products or surface-tagged fluorescent products of interest. However, traditional FC and FACS methods cannot identify or isolate cells that secrete extracellular products of interest. Double emulsion (DE) droplets are an innovative approach to retaining these extracellular products so cells producing them can be identified and isolated with FC and FACS. The water-in-oil-in-water structure makes DE droplets compatible with the sheath flow of flow cytometry. Single cells can be encapsulated with other reagents into DEs, which act as pico-reactors. These droplets allow biological activities to take place while allowing for cell cultivation monitoring, rare mutant identification, and cellular events characterization. However, using DEs in FACS presents technical challenges, including rupture of DEs, poor accuracy and low sorting efficiency. This study presents high-performance sorting using fluorescent beads (as simulants for cells). This study aims to guide researchers in the use of DE-based flow cytometry, offering insights into how to resolve the technical difficulties associated with DE-based screening and sorting using FC.

Screening, isolation, and activity evaluation of potential xanthine oxidase inhibitors in Poria Cum Radix Pini and mechanism of action in the treatment of gout disease.

Poria Cum Radix Pini is a rare medicinal fungus that contains several potential therapeutic ingredients. On this basis, a particle swarm mathematical model was used to optimize the extraction process of total triterpenes from P. Cum Radix Pini, and xanthine oxidase inhibitors were screened using affinity ultrafiltration mass spectrometry. Meanwhile, the accuracy of the ultrafiltration assay was verified by molecular docking experiments and molecular dynamics analysis, and the mechanism of action of the active compounds for the treatment of gout was analyzed by enzymatic reaction kinetics and network pharmacology. A high-speed countercurrent chromatography method combined with the consecutive injection and the economical two-phase solvent system preparation using functional activity coefficient of universal quasichemical model (UNIFAC) mathematical model was developed for increasing the yield of target compound. In addition, dehydropachymic acid and pachymic acid were used as competitive inhibitors, and 3-O-acetyl-16alpha-hydroxydehydrotrametenolic acid and dehydrotrametenolic acid were used as mixed inhibitors. Then, activity-oriented separation and purification were performed by high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography and the purity of the four compounds isolated was higher than 90%. It will help to provide more opportunities to discover and develop new potential therapeutic remedies from health care food resources.

Rapid screening, isolation, and activity evaluation of potential xanthine oxidase inhibitors in Polyporus umbellatus and mechanism of action in the treatment of gout.

INTRODUCTION: Studies show that Polyporus umbellatus has some pharmacological effects in enhancing immunity and against gout. OBJECTIVES: We aimed to establish new techniques for extraction, biological activity screening, and preparation of xanthine oxidase inhibitors (XODIs) from P. umbellatus. METHODS: First, the extraction of P. umbellatus was investigated using the back propagation (BP) neural network genetic algorithm mathematical regression model, and the extraction variables were optimised to maximise P. umbellatus yield. Second, XODIs were rapidly screened using ultrafiltration, and the change of XOD activity was tested by enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on XOD. Meanwhile, the potential anti-gout effects of the obtained active substances were verified using molecular docking, molecular dynamics simulations, and network pharmacology analysis. Finally, with activity screening as guide, a high-speed countercurrent chromatography (HSCCC) method combined with consecutive injection and two-phase solvent system preparation using the UNIFAC mathematical model was successfully developed for separation and purification of XODIs, and the XODIs were identified using MS and NMR. RESULTS: The results verified that polyporusterone A, polyporusterone B, ergosta-4,6,8(14),22-tetraen-3-one, and ergosta-7,22-dien-3-one of P. umbellatus exhibited high biological affinity towards XOD. Their structures have been further identified by NMR, indicating that the method is effective and applicable for rapid screening and identification of XODIs. CONCLUSION: This study provides new ideas for the search for natural XODIs active ingredients, and the study provide valuable support for the further development of functional foods with potential therapeutic benefits.

Tuneable Cell-Laden Double-Emulsion Droplets for Enhanced Signal Detection.

Water-in-oil-in-water (w/o/w) or double-emulsion (DE) droplets have been widely used for cellular assays at a single-cell level because of their stability and biocompatibility. The oil shell of w/o/w droplets plays the role of a semipermeable membrane that allows substances with low molecular weight (e.g., water) to travel through but restricts those with high molecular weight (e.g., fluorescent biomarkers). Therefore, the core of DEs can be manipulated using osmosis, resulting in the shrinking or swelling of the core. Water leaves the inner aqueous phase to the outer phase via the oil shell when the osmotic pressure of the outer phase is higher than that in the inner phase, causing the shrinkage of DEs and vice versa. These processes can be achieved by transferring the DEs to hypertonic or hypotonic solutions. Manipulation of the core size of DEs can be beneficial to cellular assays. First, due to the selectivity of the oil shell of DEs, the concentration of biomarkers in the core increases when the inner aqueous phase is shrunk, resulting in the enhancement of biosignals. We demonstrate this by encapsulating the Bgl3 enzyme-secreting yeast with a substrate that displays fluorescence after hydrolyzation. In a second application, a single GFP-tagged yeast cell was encapsulated in DEs. After swelling the core of DEs, we observe that the larger core of DEs promotes cell growth compared to those with the smaller cores, leading to more intracellular proteins (green-fluorescent protein) for screening. These osmotic manipulations provide new tools for droplet-based biochemistry.

Extraction and preparation of 5-lipoxygenase and acetylcholinesterase inhibitors from Astragalus membranaceus stems and leaves.

In this study, an efficient method that employs 5-lipoxygenase and acetylcholinesterase as biological target molecules in receptor-ligand affinity ultrafiltration-liquid chromatography was developed for the screening of enzyme inhibitors derived from the Astragalus membranaceus stems and leaves. The effects of the extraction time, number of extraction cycles, ethanol concentration, and liquid-solid ratio on the total yield of the target compounds were investigated using response surface methodology, and the bioactive components were isolated using a combination of semi-preparative high-performance liquid chromatography and high-speed countercurrent chromatography via a two-phase solvent system consisting of n-hexane-ethyl acetate-methanol-water (1:6:2:6, v/v/v/v). Subsequently, 10 naturally-occurring bioactive components in the Astragalus membranaceus stems and leaves, including wogonin, ononin, isoquercitrin, calycosin-7-glucoside, 3-hydroxy-9,10-dimethoxyptercarpan, hyperoside, 7,2'-dihydroxy-3',4'-dimethoxyisoflavan, baicalein, calycosin, and soyasaponin, were screened using affinity ultrafiltration to determine their potential effects against Alzheimer's disease. Consequently, all target compounds had purities higher than 95.0%, and the potential anti-Alzheimer's disease effect of the obtained bioactive compounds was verified using molecular docking analysis. Based on the results, the back-to-back screening of complex enzyme inhibitors and separation of the target bioactive compounds using complex chromatography could provide a new approach to the discovery and preparation of natural active ingredients.

Receptor-ligand affinity-based screening and isolation of water-soluble 5-lipoxygenase inhibitors from Phellinus igniarius.

We developed an efficient combination method for extraction, biological activity screening, and preparation of 5-lipoxygenase inhibitors from Phellinus igniarius. 5-Lipoxygenase inhibitors were rapidly screened using ultrafiltration-liquid chromatography based on the receptor-ligand affinity. Parameters such as extraction time, extraction times, and temperature as well as liquid-solid ratio were optimized using response surface methodology to maximize the total yield of the three target compounds. Next, bioactive ingredients were isolated using high-speed countercurrent chromatography and semi-preparative liquid chromatography. Three active ingredients, phellibaumin E, protocatechuic aldehyde, and osmundacetone, were obtained via ultrafiltration-liquid chromatography. Subsequently, the potential anti-dementia effects of the obtained bioactive compounds were verified using molecular docking assays. The above-mentioned target compounds, with purities of 98.82%, 98.89%, and 99.51%, respectively, were separated using a two-phase solvent system consisting of n-hexane-ethyl acetate-ethanol-water (2.5:2:0.75:3, v/v/v/v) coupled with semi-preparative liquid chromatography.

Comparison of flexible fixation and screw fixation for isolated Lisfranc ligament injuries: A protocol for a meta-analysis of comparative studies.

BACKGROUND: The frequency of isolated Lisfranc ligament (ILL) injuries has been increasing recently with the increase in low-energy trauma resulting from sports injuries. For ILL injuries, the optimal method of fixation still remains controversial. The traditional fixation method is achieved by trans-articular screws, but recently, dorsal bridge plates and suture button (SB) fixation have become alternatives. Some biomechanical studies have showed that SB fixation can provide adequate strength compared to trans-articular screws. Therefore, a meta-analysis is imperative to provide evidence on whether flexible fixation is comparable to screw fixation for treatment of ILL injuries. METHODS: We will conduct a comprehensive literature search in PubMed, Cochrane Library, EMBASE and Web of Science databases and for comparative studies. We will apply the risk-of-bias tool of the Cochrane Collaboration for Randomized Controlled Trials to assess the methodological quality. Risk-of-Bias Assessment Tool for Non-randomized Studies was used to evaluate the quality of comparative studies. Statistical analysis will be conducted using RevMan 5.4 software (Cochrane Collaboration, London, England). RESULTS: This systematic review will evaluate the functional outcomes and radiographic results of flexible fixation for treatment of ILL injuries. CONCLUSION: The conclusion of this study will provide evidence for judging whether flexible fixation is superior to screw fixation for treatment of ILL injuries.

Droplet flow cytometry for single-cell analysis.

The interrogation of single cells has revolutionised biology and medicine by providing crucial unparalleled insights into cell-to-cell heterogeneity. Flow cytometry (including fluorescence-activated cell sorting) is one of the most versatile and high-throughput approaches for single-cell analysis by detecting multiple fluorescence parameters of individual cells in aqueous suspension as they flow past through a focus of excitation lasers. However, this approach relies on the expression of cell surface and intracellular biomarkers, which inevitably lacks spatial and temporal phenotypes and activities of cells, such as secreted proteins, extracellular metabolite production, and proliferation. Droplet microfluidics has recently emerged as a powerful tool for the encapsulation and manipulation of thousands to millions of individual cells within pico-litre microdroplets. Integrating flow cytometry with microdroplet architectures surrounded by aqueous solutions (e.g., water-in-oil-in-water (W/O/W) double emulsion and hydrogel droplets) opens avenues for new cellular assays linking cell phenotypes to genotypes at the single-cell level. In this review, we discuss the capabilities and applications of droplet flow cytometry (DFC). This unique technique uses standard commercially available flow cytometry instruments to characterise or select individual microdroplets containing single cells of interest. We explore current challenges associated with DFC and present our visions for future development.