Sinomenine Hydrochloride Protects against Polymicrobial Sepsis via Autophagy

Sepsis, a systemic inflammatory response to infection, is the major cause of death in intensive care units (ICUs). The mortality rate of sepsis remains high even though the treatment and understanding of sepsis both continue to improve. Sinomenine (SIN) is a natural alkaloid extracted from Chinese medicinal plant Sinomenium acutum, and its hydrochloride salt (Sinomenine hydrochloride, SIN-HCl) is widely used to treat rheumatoid arthritis (RA). However, its role in sepsis remains unclear. In the present study, we investigated the role of SIN-HCl in sepsis induced by cecal ligation and puncture (CLP) in BALB/c mice and the corresponding mechanism. SIN-HCl treatment improved the survival of BALB/c mice that were subjected to CLP and reduced multiple organ dysfunction and the release of systemic inflammatory mediators. Autophagy activities were examined using Western blotting. The results showed that CLP-induced autophagy was elevated, and SIN-HCl treatment further strengthened the autophagy activity. Autophagy blocker 3-methyladenine (3-MA) was used to investigate the mechanism of SIN-HCl in vitro. Autophagy activities were determined by examining the autophagosome formation, which was shown as microtubule-associated protein light chain 3 (LC3) puncta with green immunofluorescence. SIN-HCl reduced lipopolysaccharide (LPS)-induced inflammatory cytokine release and increased autophagy in peritoneal macrophages (PM). 3-MA significantly decreased autophagosome formation induced by LPS and SIN-HCl. The decrease of inflammatory cytokines caused by SIN-HCl was partially aggravated by 3-MA treatment. Taken together, our results indicated that SIN-HCl could improve survival, reduce organ damage, and attenuate the release of inflammatory cytokines induced by CLP, at least in part through regulating autophagy activities.     

Bile Duct Ligation Impairs Function and Expression of Mrp1 at Rat Blood–Retinal Barrier via Bilirubin-Induced P38 MAPK Pathway Activations

Liver injury is often associated with hepatic retinopathy, resulting from accumulation of retinal toxins due to blood–retinal barrier (BRB) dysfunction. Retinal pigment epithelium highly expresses MRP1/Mrp1. We aimed to investigate whether liver injury affects the function and expression of retinal Mrp1 using bile duct ligation (BDL) rats. Retinal distributions of fluorescein and 2,4-dinitrophenyl-S-glutathione were used for assessing Mrp1 function. BDL significantly increased distributions of the two substrates and bilirubin, downregulated Mrp1 protein, and upregulated phosphorylation of p38 and MK2 in the retina. BDL neither affected the retinal distribution of FITC-dextran nor expressions of ZO-1 and claudin-5, demonstrating intact BRB integrity. In ARPE-19 cells, BDL rat serum or bilirubin decreased MRP1 expression and enhanced p38 and MK2 phosphorylation. Both inhibiting and silencing p38 significantly reversed the bilirubin- and anisomycin-induced decreases in MRP1 protein. Apparent permeability coefficients of fluorescein in the A-to-B direction (P_{app, A-to-B}) across the ARPE-19 monolayer were greater than P_{app, B-to-A}. MK571 or bilirubin significantly decreased P_{app, A-to-B} of fluorescein. Bilirubin treatment significantly downregulated Mrp1 function and expression without affecting integrity of BRB and increased bilirubin levels and phosphorylation of p38 and MK2 in rat retina. In conclusion, BDL downregulates the expression and function of retina Mrp1 by activating the p38 MAPK pathway due to increased bilirubin levels.     

Alkoxy Tetrazine Substitution at a Boron Center: A Strategy for Synthesizing Highly Fluorogenic Hydrophilic Probes

Strongly fluorogenic boron dipyrromethene (BODIPY)–tetrazine probes have been obtained by introducing an alkoxy tetrazine fragment at the boron center. The fluorescence signal from these probes strongly increases by up to 225‐fold after reaction with bioorthogonal coupling partners, and the hydrophilicity of probes is improved, such that they are suitable for live‐cell imaging.     

Glycometabolic Bioorthogonal Chemistry‐Guided Viral Transduction for Robust Human T Cell Engineering

Genetically engineered T cell therapy is emerging as a potent strategy for treating hematological and solid malignancies. Although lentivirus is the most common vector for T cell gene modification, its transduction efficacy remains unsatisfied especially during the manufacturing process. Herein, glycometabolic bioorthogonal chemistry is utilized to establish a highly efficient viral transduction system for human primary T lymphocytes. Azide motifs are anchored on the T cell surface via the intrinsic glycometabolism of exogenous azide–glucose, serving as an artificial ligand for viral binding. The complementary functional moiety dibenzocyclooctyl (DBCO)‐conjugated PEI_1.8K (PEI‐DBCO) is then coated on lentiviral surface, which strengthens the virus–T cell interaction through DBCO/azide bioorthogonal chemistry. The results show that the PEI‐DBCO/azide–glucose system effectively facilitates viral binding to T cells and elevates the transduction efficiency of the lentivirus from 20% to 80% without any effect on T cell proliferation and activity. More importantly, the PEI‐DBCO/azide–glucose system significantly doubles the yield of anti‐CD19 chimeric antigen receptor T (CAR‐T) cells and robustly boosts their antitumor capability compared to polybrene‐assisted lentiviral transduction both in vitro and in vivo. Overall, the bioorthogonal PEI‐DBCO/azide–glucose system significantly boosts viral transduction efficacy and exhibits a powerful gene‐manipulating capability in human primary T cells, thereby showing a great potential for clinical‐engineered T lymphocytes manufacture.     

A Sweet Galactose Transfer: Metabolic Oligosaccharide Engineering as a Tool To Study Glycans in Plasmodium Infection

The introduction of chemical reporter groups into glycan structures through metabolic oligosaccharide engineering (MOE) followed by bio-orthogonal ligation is an important tool to study glycosylation. We show the incorporation of synthetic galactose derivatives that bear terminal alkene groups in hepatic cells, with and without infection by Plasmodium berghei parasites, the causative agent of malaria. Additionally, we demonstrated the contribution of GLUT1 to the transport of these galactose derivatives, and observed a consistent increase in the uptake of these compounds going from naïve to P. berghei-infected cells. Finally, we used MOE to study the interplay between Plasmodium parasites and their mosquito hosts, to reveal a possible transfer of galactose building blocks from the latter to the former. This strategy has the potential to provide new insights into Plasmodium glycobiology as well as for the identification and characterization of key glycan structures for further vaccine development.     

Recent Developments and Strategies for Mutually Orthogonal Bioorthogonal Reactions

Over the past decade, several different metal-free bioorthogonal reactions have been developed to enable simultaneous double-click labeling with minimal-to-no competing cross-reactivities; such transformations are termed ‘mutually orthogonal’. More recently, several examples of successful triple ligation strategies have also been described. In this minireview, we discuss selected aspects of the development of orthogonal bioorthogonal reactions over the past decade, including general strategies to drive future innovations to achieve simultaneous, mutually orthogonal click reactions in one pot.