Catalyst-free thiazolidine formation chemistry enables the facile construction of peptide/protein-cell conjugates (PCCs) at physiological pH.

Although numerous genetic, chemical, and physical strategies have been developed to remodel the cell surface landscape for basic research and the development of live cell-based therapeutics, new chemical modification strategies capable of decorating cells with various genetically/non-genetically encodable molecules are still urgently needed. Herein, we describe a remarkably simple and robust chemical strategy for cell surface modifications by revisiting the classical thiazolidine formation chemistry. Cell surfaces harbouring aldehydes can be chemoselectively conjugated with molecules containing a 1,2-aminothiol moiety at physiological pH without the need to use any toxic catalysts and complicated chemical synthesis. Through the combined use of thiazolidine formation and the SpyCatcher-SpyTag system, we have further developed a SpyCatcher-SpyTag Chemistry Assisted Cell Surface Engineering (SpyCASE) platform, providing a modular approach for the construction of large protein-cell conjugates (PCCs) in their native state. Thiazolidine-bridged molecules can also be detached from the surface again through a biocompatible Pd-catalyzed bond scission reaction, enabling reversible modification of living cell surfaces. In addition, this approach allows us to modulate specific cell-cell interactions and generate NK cell-based PCCs to selectively target/kill several EGFR-positive cancer cells in vitro. Overall, this study provides an underappreciated but useful chemical tool to decorate cells with tailor-made functionalities.

Cholesterol-autoxidation metabolites in host defense against infectious diseases.

Cholesterol plays essential roles in biological processes, including cell membrane stability and myelin formation. Cholesterol can be metabolized to oxysterols by enzymatic or nonenzymatic ways. Nonenzymatic cholesterol metabolites, also called cholesterol-autoxidation metabolites, are formed dependent on the oxidation of reactive oxygen species (ROS) such as OH• or reactive nitrogen species, such as ONOO- . Cholesterol-autoxidation metabolites are abundantly produced in diseases such as inflammatory bowel disease and atherosclerosis, which are associated with oxidative stress. Recent studies have shown that cholesterol-autoxidation metabolites can further regulate the immune system. Here, we review the literature and summarize how cholesterol-autoxidation metabolites, such as 25-hydroxycholesterol (25-OHC), 7α/ß-OHC, and 7-ketocholesterol, deal with the occurrence and development of infectious diseases through pattern recognition receptors, inflammasomes, ROS production, nuclear receptors, G-protein-coupled receptor 183, and lipid availability. In addition, we include the research regarding the roles of these metabolites in COVID-19 infection and discuss our viewpoints on the future research directions.

Chemical and Biological Strategies for Profiling Protein-Protein Interactions in Living Cells.

Protein-protein interactions (PPIs) play critical roles in almost all cellular signal transduction events. Characterization of PPIs without interfering with the functions of intact cells is very important for basic biology study and drug developments. However, the ability to profile PPIs especially those weak/transient interactions in their native states remains quite challenging. To this end, many endeavors are being made in developing new methods with high efficiency and strong operability. By coupling with advanced fluorescent microscopy and mass spectroscopy techniques, these strategies not only allow us to visualize the subcellular locations and monitor the functions of protein of interest (POI) in real time, but also enable the profiling and identification of potential unknown interacting partners in high-throughput manner, which greatly facilitates the elucidation of molecular mechanisms underlying numerous pathophysiological processes. In this review, we will summarize the typical methods for PPIs identification in living cells and their principles, advantages and limitations will also be discussed in detail.

Dexmedetomidine ameliorates ischemia-induced nerve injury by up-regulating Sox11 expression.

Background: Dexmedetomidine (Dex) is associated with several biological processes. Ischemic stroke has the characteristics of high morbidity and mortality. Herein, we aimed to explore whether Dex ameliorates ischemia-induced injury and determine its mechanism. Methods: Real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting were used to measure gene and protein expression. Cellular viability and proliferation were assessed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays, respectively. Cell apoptosis was detected by flow cytometry. An oxygen-glucose deprivation/reoxygenation model of SK-N-SH and SH-SY5Y cells was constructed. A middle cerebral artery occlusion (MCAO) model was also built to assess Dex function in vivo. Neuronal function was assessed using the Bederson Behavior Score and Longa Behavior Score. Results: We found that Dex positively and dose-dependently regulated Sox11 expression and prevented damage caused by oxygen-glucose deprivation/reoxygenation (OGD/R), enhancing cell viability and proliferation and reducing apoptosis in SK-N-SH and SH-SY5Y cells. The overexpression of Sox11 antagonized OGD/R-induced SK-N-SH and SH-SY5Y cell apoptosis and promoted cell growth in vitro. Furthermore, cell proliferation was decreased and cell apoptosis was increased after Sox11 knockdown in Dex-treated SK-N-SH and SH-SY5Y cells. We demonstrated that Dex prevented OGD/R-induced cell injury by up-regulating Sox11. Furthermore, we also confirmed that Dex protected rat from ischemia-induced injury in the MCAO model. Conclusions: The role of Dex in cell viability and survival was verified in this study. Moreover, Dex protected neurons from MCAO-induced injury by up-regulating the expression of Sox11. Our research proposes a potential drug to improve the functional recovery of stroke patients in the clinic.

PAL-Mediated Ligation for Protein and Cell-Surface Modification.

Peptidyl Asx-specific ligases (PALs) effect peptide ligation by catalyzing transpeptidation reactions at Asn/Asp-peptide bonds. Owing to their high efficiency and mild aqueous reaction conditions, these ligases have emerged as powerful biotechnological tools for protein manipulation in recent years. PALs are enzymes of the asparaginyl endopeptidase (AEP) superfamily but have predominant transpeptidase activity as opposed to typical AEPs which are predominantly hydrolases. Butelase-1 and VyPAL2, two PALs discovered by our teams, have been used successfully in a wide range of applications, including macrocyclization of synthetic peptides and recombinant proteins, protein N- or C-terminal modification, and cell-surface labeling. As shown in numerous reports, PAL-mediated ligation is highly efficient at Asn junctions. Although considerably less efficient, Asp-specific ligation has also been shown to be practically useful under suitable conditions. Herein, we describe the methods of using VyPAL2 for protein macrocyclization and labeling at an Asp residue as well as for protein dual labeling through orthogonal Asp- and Asn-directed ligations. We also describe a method for cell-surface protein modification using butelase-1, demonstrating its advantageous features over previous methods.

[Retracted] miR­218 suppresses tumor growth and enhances the chemosensitivity of esophageal squamous cell carcinoma to cisplatin.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that certain of the western blotting data shown in Fig. 6 and the tumor images shown in Fig. 7A were strikingly similar to data appearing in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they agreed with the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in Oncology Reports 33: 981­989, 2015; DOI: 10.3892/or.2014.3657].

Tagging Transferrin Receptor with a Disulfide FRET Probe To Gauge the Redox State in Endosomal Compartments.

Although the basic process of receptor-mediated endocytosis (RME) is well established, certain specific aspects, like the endosomal redox state, remain less characterized. Previous studies used chemically labeled ligands or antibodies with a FRET (fluorescence resonance energy transfer) probe to gauge the redox activity of the endocytic pathway with a limitation being their inability to track the apo receptor. New tools that allow direct labeling of a cell surface receptor with synthetic probes would aid in the study of its endocytic pathway and function. Herein, we use a peptide ligase, butelase 1, to label the human transferrin receptor 1 (TfR1) in established human cell lines with a designer disulfide FRET probe. This strategy enables us to obtain real-time live cell imaging of redox states in TfR1-mediated endocytosis, attesting a reducing environment in the endosomal compartments and the dynamics of TfR1 trafficking. A better understanding of endocytosis of different cell surface receptors has implications in designing strategies that hijack this natural process for intracellular drug delivery.

A review of the phytochemistry and pharmacological activities of Ephedra herb.

Ephedra herb is a traditional Chinese medicine with a long history. Conventionally, it was used as a folk phytomedicine in many ancient medical books and traditional prescriptions. Up to date, a variety of specific ingredients have been found in Ephedra herb, mainly including alkaloids, flavonoids, tannins, polysaccharides, organic acids, volatile oils, and many other active compounds. These components from Ephedra herb account for its use as the accurate treatment of cold, cough, cardiovascular and immune system disease, cancer, microbial infection, and other diseases. Moreover, with the fast development of novel chemistry and medicine technology, new chemical constituents and pharmacological effects of Ephedra herb are increasingly identified, demonstrating their great potential for various diseases treatment. Therefore, further detailed understanding and investigation of this ancient herb will offer new opportunities to develop novel therapeutics. This study systematically reviews its progress of phytochemistry, traditional and modern pharmacology based on research data that have been reported, aiming at providing useful insight for commercial exploitation, further study and precision medication of Ephedra herb in future.

Dexmedetomidine versus sufentanil with high- or low-concentration ropivacaine for labor epidural analgesia: A randomized trial.

AIM: To study analgesic effects of dexmedetomidine or sufentanil, both combined with ropivacaine, in epidural analgesia during labor. METHODS: We recruited 160 primigravidae with full-term pregnancy who received epidural anesthesia during labor and randomized them into four groups to receive epidural administration of ropivacaine combined with sufentanil (RS1 and RS2 groups) or with dexmedetomidine (RD1 and RD2 groups). Systolic blood pressure, diastolic blood pressure and heart rate before anesthesia (T1 ), 15 min after anesthesia induction (T2 ), on delivery (T3 ) and 2 h postpartum (T4 ), together with visual analogue scale scores, Bromage scores, Ramsay scores, adverse reactions during analgesia and urinary retention at 6 and 24 h postpartum were recorded; the pH, PCO2 and PO2 of umbilical cord arterial blood and Apgar scores at 1, 5 and 10 min after childbirth were assessed. RESULTS: RS1 group had significantly lower systolic blood pressure, diastolic blood pressure and heart rate than RD1 group at T2 and T3 (all P < 0.05), but not at T1. At T2 and T3 , the other three groups were lower than RS2 group in visual analogue scale and Ramsay scores (all P < 0.05). After childbirth, RD2 group had significantly higher PO2 result than other three groups (P < 0.05). At 6 h postpartum, RD2 group had significantly fewer cases of urinary retention than RD1 and RS1 groups (both P < 0.05). CONCLUSION: A relatively low concentration of ropivacaine, combined with dexmedetomidine, is better in analgesia during labor.

Dexmedetomidine protects PC12 cells from lidocaine-induced cytotoxicity via downregulation of Stathmin 1.

Background: Understanding of lidocaine-induced neurotoxicity is not complete, resulting in the unsuccessful treatment in some clinical settings. Dexmedetomidine (DEX) has been shown to alleviate lidocaine-induced neurotoxicity in our previous cell model. However, the rationale for DEX combined with lidocaine to reduce lidocaine-induced neurotoxicity in the clinical setting remains to be further clarified in the detailed molecular mechanism. Methods: In this study, we established a cellular injury model by lidocaine preconditioning. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assay kit were used to analyze cell proliferation. Cell apoptosis was measured by flow cytometry and Hoechst 33342 staining. Cell cycle progression was detected by flow cytometry. The protein expression levels were detected by Western blotting and immunofluorescence staining. Results: Our results showed that DEX dose-dependently restored impaired proliferation of PC12 cells induced by lidocaine，as reflected by the increased cell viability and EdU positive cells, which were consistent with the decreased expression of tumor suppressor protein p21 and increased expression of cell cycle-related cyclin D1 and CDK1. In addition, DEX dose-dependently reduced apoptotic PC12 cells induced by lidocaine，as reflected by the decreased expression of apoptosis-related Bax, caspase-3 and caspase-9 and increased expression of anti-apoptotic Bcl-2 compared to the cells only treated with lidocaine. Mechanistically, with gain-or-loss-of-function of STMN1, we showed that DEX-mediated neuroprotection by lidocaine-induced damage is associated with downregulation of STMN1 which might be an upstream molecule involved in regulation of mitochondria death pathway. Conclusion: Our results reveal that DEX is likely to be an effective adjunct to alleviate chronic neurotoxicity induced by lidocaine.

Butelase 1-Mediated Ligation of Peptides and Proteins.

Structurally, butelase 1 is a cysteine protease of the asparaginyl endoprotease (AEP) family, but functionally, it displays intense Asn/Asp-specific (Asx) ligase activity and is virtually devoid of protease activity. Butelase 1 recognizes specifically a C-terminal Asx-containing tripeptide motif, Asx-His-Val, to form an Asx-Xaa peptide bond (Xaa = any amino acid), either intramolecularly or intermolecularly, resulting in cyclic peptides or site-specific modified peptides/proteins, respectively. Our work in the past 4 years has validated that butelase 1 is a potent and versatile tool for peptide and protein modification. Here we describe our protocols using butelase 1 for efficient and site-specific peptide and protein ligation, N-terminal labeling, preparation of thioesters, and bioconjugation of dendrimers. Additionally, we provide an example using butelase 1 for protein cyclization in combination with genetic code expansion in order to incorporate unnatural building blocks.

Thiazolidin-5-imine Formation as a Catalyst-Free Bioorthogonal Reaction for Protein and Live Cell Labeling.

A previously undescribed reaction involving the formation of a thiazolidin-5-imine linkage was developed for bioconjugation. Being highly specific and operating in aqueous media, this simple condensation reaction is used to chemoselectively label peptides, proteins, and living cells under physiological conditions without the need to use toxic catalysts or reducing reagents.

Immobilization and Intracellular Delivery of Circular Proteins by Modifying a Genetically Incorporated Unnatural Amino Acid.

Backbone-cyclic proteins are of great scientific and therapeutic interest owing to their higher stability over their linear counterparts. Modification of such cyclic proteins at a selected site would further enhance their versatility. Here we report a chemoenzymatic strategy to engineer site-selectively modified cyclic proteins by combining butelase-mediated macrocyclization with the genetic code expansion methodology. Using this strategy, we prepared a cyclic protein which was modified with biotin or a cell-penetrating peptide at a genetically incorporated noncanonical amino acid, making the cyclization-stabilized protein further amenable for site-specific immobilization and intracellular delivery. Our results point to a new avenue to engineering novel cyclic proteins with improved physicochemical and pharmacological properties for potential applications in biotechnology and medicine.

Chemical and Enzymatic Strategies for Bacterial and Mammalian Cell Surface Engineering.

The cell surface serves important functions such as the regulation of cell-cell and cell-environment interactions. The understanding and manipulation of the cell surface is important for a wide range of fundamental studies of cellular behavior and for biotechnological and medical applications. With the rapid advance of biology, chemistry and materials science, many strategies have been developed for the functionalization of bacterial and mammalian cell surfaces. Here, we review the recent development of chemical and enzymatic approaches to cell surface engineering with particular emphasis on discussing the advantages and limitations of each of these strategies.

Role of remodeling and spacing factor 1 in histone H2A ubiquitination-mediated gene silencing.

Posttranslational histone modifications play important roles in regulating chromatin-based nuclear processes. Histone H2AK119 ubiquitination (H2Aub) is a prevalent modification and has been primarily linked to gene silencing. However, the underlying mechanism remains largely obscure. Here we report the identification of RSF1 (remodeling and spacing factor 1), a subunit of the RSF complex, as a H2Aub binding protein, which mediates the gene-silencing function of this histone modification. RSF1 associates specifically with H2Aub, but not H2Bub nucleosomes, through a previously uncharacterized and obligatory region designated as ubiquitinated H2A binding domain. In human and mouse cells, genes regulated by RSF1 overlap significantly with those controlled by RNF2/Ring1B, the subunit of Polycomb repressive complex 1 (PRC1) which catalyzes the ubiquitination of H2AK119. About 82% of H2Aub-enriched genes, including the classic PRC1 target Hox genes, are bound by RSF1 around their transcription start sites. Depletion of H2Aub levels by Ring1B knockout results in a significant reduction of RSF1 binding. In contrast, RSF1 knockout does not affect RNF2/Ring1B or H2Aub levels but leads to derepression of H2Aub target genes, accompanied by changes in H2Aub chromatin organization and release of linker histone H1. The action of RSF1 in H2Aub-mediated gene silencing is further demonstrated by chromatin-based in vitro transcription. Finally, RSF1 and Ring1 act cooperatively to regulate mesodermal cell specification and gastrulation during Xenopus early embryonic development. Taken together, these data identify RSF1 as a H2Aub reader that contributes to H2Aub-mediated gene silencing by maintaining a stable nucleosome pattern at promoter regions.

Dexmedetomidine inhibits activation of the MAPK pathway and protects PC12 and NG108-15 cells from lidocaine-induced cytotoxicity at its maximum safe dose.

The developing brains of pediatric patients are highly vulnerable to anesthetic regimen (e.g., lidocaine), potentially causing neurological impairment. Recently, dexmedetomidine (DEX) has been used as an adjunct for sedation, and was shown to exert dose-dependent neuroprotective effects during brain injury. However, the maximum safe dose of DEX is unclear, and its protective effects against lidocaine-related neurotoxicity need to be confirmed. In this study, PC12 and NG108-15 cells were used to estimate safe, non-cytotoxic doses of DEX. We found that 100 and 60µM are the maximum safe dose of DEX for PC12 and NG108-15 cells, respectively, with no significant cytotoxicity. Lidocaine was found to remarkably inhibit cell vitality, but could be reversed by different doses of DEX, especially its maximum safe dose. Furthermore, the apoptosis induced by lidocaine was also assessed, and 100 and 60µM DEX showed optimal protective effects in PC12 and NG108-15 cells, respectively. Mechanistically, DEX activated the mitogen-activated protein kinase (MAPK) pathway, impaired caspase-3 expression, and enhanced anti-apoptotic factor Bcl-2 to resist lidocaine-induced apoptosis, indicating that the optimal dose of DEX alleviates lidocaine-induced cytotoxicity and should be considered in clinical application.

Enzymatic Engineering of Live Bacterial Cell Surfaces Using Butelase†1.

Butelase-mediated ligation (BML) can be used to modify live bacterial cell surfaces with diverse cargo molecules. Surface-displayed butelase recognition motif NHV was first introduced at the C-terminal end of the anchoring protein OmpA on E. coli cells. This then served as a handle of BML for the functionalization of E. coli cell surfaces with fluorescein and biotin tags, a tumor-associated monoglycosylated peptide, and mCherry protein. The cell-surface ligation reaction was achieved at low concentrations of butelase and the labeling substrates. Furthermore, the fluorescein-labeled bacterial cells were used to show the interactions with cultured HeLa cells and with macrophages in live transgenic zebrafish, capturing the latter's powerful phagocytic effect in action. Together these results highlight the usefulness of butelase 1 in live bacterial cell surface engineering for novel applications.

Dexmedetomidine Protects PC12 Cells from Lidocaine-Induced Cytotoxicity Through Downregulation of COL3A1 Mediated by miR-let-7b.

Safety concerns of some local anesthetics, such as lidocaine, have been raised in recent years due to potential neurological impairment. Dexmedetomidine may protect humans from neurotoxicity, and miR-let-7b is activated by nerve injury; however, the roles of miR-let-7b and its target gene in lidocaine-induced cytotoxicity are not well known. Through bioinformatics and a luciferase reporter assay, COL3A1 was suggested as a direct target gene of miR-let-7b. Here, we confirmed by measuring mRNA and protein levels that miR-let-7b was downregulated and COL3A1 was upregulated in lidocaine-treated cells, an observation that was reversed by dexmedetomidine. Similar to miR-let-7b mimics or knockdown of COL3A1, dexmedetomidine treatment reduced the expression of COL3A1, suppressed cell apoptosis and cell migration/invasion ability, and induced cell cycle progression and cell proliferation in PC12 cells, effects that were reversed by the miR-let-7b inhibitor. Meanwhile, proteins involved in cell apoptosis, such as Bcl2 and caspase 3, were impacted as well. Taken together, dexmedetomidine may protect PC12 cells from lidocaine-induced cytotoxicity through miR-let-7b and COL3A1, while also increasing Bcl2 and inhibiting caspase 3. Therefore, miR-let-7b and COL3A1 might play critical roles in neuronal injury, and they are potential therapeutic targets.

Thiazolidine-Masked α-Oxo Aldehyde Functionality for Peptide and Protein Modification.

α-Oxo aldehyde-based bioconjugation chemistry has been widely explored in peptide and protein modifications for various applications in biomedical research during the past decades. The generation of α-oxo aldehyde via sodium periodate oxidation is usually limited to the N-terminus of a target protein. Internal-site functionalization of proteins with the α-oxo aldehyde handle has not been achieved yet. Herein we report a novel method for site-specific peptide and protein modification using synthetically or genetically incorporated thiazolidine-protected α-oxo aldehyde. Efficient unmasking of the aldehyde was achieved by silver ion-mediated hydrolysis of thiazolidine under mild conditions for the first time. A model peptide and a recombinant protein were used to demonstrate the utility of this new method, which were site-specifically modified by oxime ligation with an oxyamine-functionalized peptide labeling reagent. Therefore, our current method has enriched the α-oxo aldehyde synthetic tool box in peptide and protein bioconjugation chemistry and holds great potential to be explored in novel applications in the future.

Genetic incorporation of 1,2-aminothiol functionality for site-specific protein modification via thiazolidine formation.

Here we report a new site-specific conjugation strategy to modify proteins via thiazolidine ligation. Proteins harbouring a 1,2-aminothiol moiety introduced by amber codon suppression technology could be modified chemoselectively with aldehyde-functionalized reagents, such as a biotin-labeled peptide or ubiquitin, under mild conditions to yield homogeneous biotinylated or ubiquitinated products.