Ratiometric imaging of butyrylcholinesterase activity in mice with nonalcoholic fatty liver using an AIE-based fluorescent probe.

Butyrylcholinesterase (BChE) is an essential human biomarker which is related to liver and neurodegenerative diseases. It is of great significance to develop a fluorescent probe that can image BChE in vitro and in vivo. Unfortunately, most fluorescent probes that are based on a single change in fluorescence intensity are susceptible to environmental interference. Therefore, we reported an easily available ratiometric fluorescent probe, TB-BChE, with aggregation-induced emission (AIE) characteristics for ratiometric imaging of BChE. TB-BChE demonstrated excellent sensitivity (LOD = 39.24 ng mL-1) and specificity for BChE. Moreover, we have successfully studied the ratiometric imaging of TB-BChE to BChE in a nonalcoholic fatty liver disease model. These results indicated that TB-BChE is expected to become a powerful analysis tool for butyrylcholinesterase research in basic medicine and clinical applications.

An easily available endoplasmic reticulum targeting near-infrared fluorescent probe for esterase imaging in vitro and in vivo.

Here, we report an easily available endoplasmic reticulum-targeting near-infrared fluorescent probe (ER-CE), which can detect esterase in the endoplasmic reticulum and monitor the changes in the esterase amount in tumors in mice in real time. These results indicate that ER-CE is expected to become a powerful analysis tool for the research of endoplasmic reticulum esterase-related diseases.

Ultrasensitive electrochemical assay for microRNA-21 based on CRISPR/Cas13a-assisted catalytic hairpin assembly.

MicroRNAs (miRNAs) are related to many biological processes and regarded as biomarkers of disease. Rapid, sensitive, and specific methods for miRNA assay are very important for early disease diagnostic and therapy. In the present work, an ultrasensitive electrochemical biosensing platform has been developed for miRNA-21 assay by combining CRISPR-Cas13a system and catalytic hairpin assembly (CHA). In the presence of miRNA-21, it would hybridize with the spacer region of Cas13a/crRNA duplex to activate the cleavage activity of CRISPR-Cas13a system, leading to the release of initiator of CHA to generate amplified electrochemical signals. Base on the CRISPR-Cas13a-mediated cascade signal amplification strategy, the developed electrochemical biosensing platform exhibited high sensitivity with a low detection limit of 2.6 fM (S/N = 3), indicating that the platform has great potential for application in early clinical diagnostic.

Preparation of TiO2/Bi/Fe/Zr nanocomposite for the highly selective enrichment of phosphopeptides.

It is necessary to effectively absorb trace phosphopeptides/proteins from complex phosphopeptide-containing samples for mass spectrometry (MS)-based phosphoproteomics. Here, Bi3+/Fe3+/Zr4+co-doped TiO2 (TiO2/Bi/Fe/Zr) nanocomposite with a high surface-to-volume ratio was synthesized by sol-gel method for selective enrichment of phosphopeptides. The enrichment ability of this modified TiO2 toward phosphopeptides was evaluated by using standard phosphoproteins and HeLa cell lysate. The results demonstrated that, compared with commercial titanium dioxide for phosphopeptides enrichment, this proposed TiO2/Bi/Fe/Zr nanocomposite has a lower detection limit (4 × 10-10 M) and higher selectivity at a low weight ratio of phosphopeptides/nonphosphopeptides (1:1000). Additionally, a total of 676 phosphorylation sites were identified from the HeLa cell lysate after enrichment by the TiO2/Bi/Fe/Zr nanocomposite. In brief, the TiO2/Bi/Fe/Zr nanocomposite exhibits better performance on the selectively absorption of phosphopeptides from either simple or complex biological samples, indicating the great potential application in phosphoproteomics study.

Quantitative phosphoproteomics of lectin receptor-like kinase VI.4 dependent abscisic acid response in Arabidopsis thaliana.

Lectin receptor-like kinases (LecRKs) play important roles in the responses to adverse environment stress. Abscisic acid (ABA) is a plant hormone involved in plant growth, development and adverse environmental stress responses. Although some studies of ABA response LecRK genes have been reported, the molecular mechanisms of LecRKs regulation of downstream pathways under ABA induction are not well understood. The present study showed that LecRK-VI.4 responded to ABA and negatively regulated stomatal closure. Here, a quantitative phosphoproteomics approach based on mass spectrometry was employed to study the roles of LecRK-VI.4 in the ABA signaling pathway. Metal oxide affinity beads and C18 chromatography were used for phosphopeptide enrichment and separation. The isobaric tags for relative and absolute quantitation were used for profiling the phosphoproteome of mutant lecrk-vi.4-1 and wild-type Col-0 Arabidopsis under normal growth conditions or ABA treatments. In total, 475 unique phosphopeptides were quantified, including 81 phosphopeptides related to LecRK-VI.4 regulation. Gene ontology, protein-protein interaction and motif analysis were performed. The bioinformatics data showed that phosphorylated proteins regulated by LecRK-VI.4 had close relations with factors of stomatal function, which included aquaporin activity, H+ pump activity and the Ca2+ concentration in the cytoplasm. These data have expanded our understanding of how LecRK-VI.4 regulates ABA-mediated stomatal movements.

Preparation of Bi0.15Fe0.15TiO2 Nanocomposites for the Highly Selective Enrichment of Phosphopeptides.

Novel Bi0.15Fe0.15TiO2 nanocomposites (B0.15F0.15TNs) were synthesized for the first time by a modified sol-gel technology and successfully applied to selective extraction and enrichment of phosphopeptides from digested protein mixture solutions and real samples (tissue protein extract from human liver). The codoping of Bi and Fe into TiO2 results in a significant enhancement in both the enrichment efficiency and selectivity. Compared with the commercial available TiO2 extractant, the proposed B0.15F0.15TNs possess a lower detection limit (2 × 10-9 M) and higher selectivity at a low weight ratio of 1:1200 (phosphopeptides/nonphosphopeptides). Additionally, a total of 223 phosphorylation sites were identified from the human liver lysate after enrichment by the B0.15F0.15TNs. In addition, the synthesis of B0.15F0.15TNs is quite easy, of high yield, and inexpensive.

Arginine modification of lycosin-I to improve inhibitory activity against cancer cells.

Lycosin-I is a linear amphipathic α-helical anticancer peptide (ACP) extracted from the spider Lycosa singoriensis, which can activate the mitochondrial death pathway to induce apoptosis in tumor cells and up-regulate p27 to inhibit cell proliferation. However, the applicability of lycosin-I as a novel anticancer drug is limited by its low cellular entry and efficacy in solid tumors. Amino acid substitution presents an effective and modest strategy to improve the anticancer activity and bioavailability of ACPs. Herein, an arginine-modified lycosin-I (named R-lycosin-I) was designed and synthesized by substituting lysine (Lys) with arginine (Arg). This peptide exhibited higher anticancer activity and penetrability against solid tumor cells than lycosin-I. They displayed noticeable differences in their physicochemical properties including the secondary structure, hydrodynamic size, and zeta potential. Fluorescence analyses have confirmed that R-lycosin-I exhibits increased cellular uptake and improved intracellular distribution. Due to its superior physical and chemical properties and high serum stability, R-lycosin-I could penetrate deeply into tumor spheroids and produce strong toxicity in the 3D tumor model. Overall, these findings suggest that arginine modification may provide an effective strategy for improving the anticancer activity of lycosin-I, and R-lycosin-I may be a useful lead for developing anticancer drugs.