Selenol Switch Assay for Selenoprotein Derivatization.

Selenoproteins are a class of protein that have selenocysteine (Sec) residues, and essential for diverse cellular functions. Although the human genome encodes 25 selenoproteins, nearly half of these selenoproteins' function is not clear. This is largely due to the lack of convenient methods to study selenoproteins. We report in this work a novel Selenol Switch assay to exclusively derivatize selenoproteins. The Selenol Switch assay relies on the selective conversion of the Sec residue to the electrophilic dehydroalanine (DHA) residue, which is then labeled by nucleophiles. The multiple reactions of the Selenol Switch assay are readily performed in a single test tube, and the conversion yield is nearly quantitative. The abundance of selenoproteins in mouse tissues determined by the Selenol Switch assay is consistent with that from the classical ICP-MS assay, validating the reliability of the Selenol Switch assay in studying selenoproteins.

Imaging of Carbonic Anhydrase Level in Epilepsy with an Environment-Sensitive Fluorescent Probe.

Carbonic anhydrases (CAs) participate in various physiological and pathological activities by catalyzing the interconversion between carbon dioxide and bicarbonate ions. Under normal circumstances, they guarantee that the relevant biological reactions in our body occur within an appropriate time scale. Abnormal expression or activity alteration of CAs is closely related to the pathogenesis of diverse diseases. This work reports an inhibitor-directed fluorescent probe FMRs-CA for the detection of CAs. Excellent selectivity, favorable biocompatibility, and desirable blood-brain barrier (BBB) penetration endow the probe with the ability to image the fluctuation of CAs in cells and mice. We achieved in situ visualization of the increased CAs in hypoxic cells with this probe. Additionally, probe FMRs-CA was mainly enriched within the liver and gradually metabolized by the liver. With the help of FMRs-CA, the increase of CAs in epileptic mouse brains was revealed first from the perspective of imaging, providing the mechanism connection between abnormal CA expressions and epilepsy.

Well-Paired-Seq: A Size-Exclusion and Locally Quasi-Static Hydrodynamic Microwell Chip for Single-Cell RNA-Seq.

Single-cell RNA sequencing (scRNA-seq) is a powerful technology for revealing the heterogeneity of cellular states. However, existing scRNA-seq platforms that utilize bead-based technologies suffer from a large number of empty microreactors and a low cell/bead capture efficiency. Here, Well-paired-seq is presented, which consists of thousands of size exclusion and quasi-static hydrodynamic dual wells to address these limitations. The size-exclusion principle allows one cell and one bead to be trapped in the bottom well (cell-capture-well) and the top well (bead-capture-well), respectively, while the quasi-static hydrodynamic principle ensures that the trapped cells are difficult to escape from cell-capture-wells, achieving cumulative capture of cells and effective buffer exchange. By the integration of quasi-static hydrodynamic and size-exclusion principles, the dual wells ensure single cells/beads pairing with high density, achieving excellent efficiency of cell capture (≈91%), cell/bead pairing (≈82%), and cell-free RNA removal. The high utilization of microreactors and single cells/beads enable to achieve a high throughput (≈105 cells) with low collision rates. The technical performance of Well-paired-seq is demonstrated by collecting transcriptome data from around 200 000 cells across 21 samples, successfully revealing the heterogeneity of single cells and showing the wide applicability of Well-paired-seq for basic and clinical research.