Fast Metabolic Glycan Labeling for Click-Labeling and Imaging of Sialoglycans in Living Acute Brain Slices from Mice and Human Patients.

Living acute brain slices provide a practical platform for imaging sialylation in human brain pathology. However, the limited lifespan of acute brain slices has impeded the use of metabolic glycan labeling (MGL), which requires long-term incubation of clickable unnatural sugars such as N-azidoacetylmannosamine (ManNAz) to metabolically incorporate azides into sialoglycans. Here, we report a fast variant of MGL (fMGL), in which ManNAz-6-phosphate enables efficient azidosugar incorporation within 12 h by bypassing the bottleneck step in the sialic acid biosynthesis pathway, followed by click-labeling with fluorophores and imaging of sialoglycans in acute brain slices from mice and human patients. In the clinical samples of ganglioglioma, fMGL-based imaging reveals specific upregulation of sialylation in astrocyte-like but not neuron-like tumor cells. In addition, fMGL is integrated with click-expansion microscopy for high-resolution imaging of sialoglycans in brain slices. The fMGL strategy should find broad applications in the tissue imaging of glycans and surgical pathology.

Click-ExM enables expansion microscopy for all biomolecules.

Expansion microscopy (ExM) allows super-resolution imaging on conventional fluorescence microscopes, but has been limited to proteins and nucleic acids. Here we develop click-ExM, which integrates click labeling into ExM to enable a 'one-stop-shop' method for nanoscale imaging of various types of biomolecule. By click labeling with biotin and staining with fluorescently labeled streptavidin, a large range of biomolecules can be imaged by the standard ExM procedure normally used for proteins. Using 18 clickable labels, we demonstrate click-ExM on lipids, glycans, proteins, DNA, RNA and small molecules. We demonstrate that click-ExM is applicable in cell culture systems and for tissue imaging. We further show that click-ExM is compatible with signal-amplification techniques and two-color imaging. Click-ExM thus provides a convenient and versatile method for super-resolution imaging, which may be routinely used for cell and tissue samples.

Cell-type-specific labeling and profiling of glycans in living mice.

Metabolic labeling of glycans with clickable unnatural sugars has enabled glycan analysis in multicellular systems. However, cell-type-specific labeling of glycans in vivo remains challenging. Here we develop genetically encoded metabolic glycan labeling (GeMGL), a cell-type-specific strategy based on a bump-and-hole pair of an unnatural sugar and its matching engineered enzyme. N-pentynylacetylglucosamine (GlcNAl) serves as a bumped analog of N-acetylglucosamine (GlcNAc) that is specifically incorporated into glycans of cells expressing a UDP-GlcNAc pyrophosphorylase mutant, AGX2F383G. GeMGL with the 1,3-di-O-propionylated GlcNAl (1,3-Pr2GlcNAl) and AGX2F383G pair was demonstrated in cell cocultures, and used for specific labeling of glycans in mouse xenograft tumors. By generating a transgenic mouse line with AGX2F383G expressed under a cardiomyocyte-specific promoter, we performed specific imaging of cardiomyocyte glycans in the heart and identified 582 cardiomyocyte O-GlcNAcylated proteins with no interference from other cardiac cell types. GeMGL will facilitate cell-type-specific glycan imaging and glycoproteomics in various tissues and disease models.

Naringin protects against inflammation and apoptosis induced by intestinal ischemia-reperfusion injury through deactivation of cGAS-STING signaling pathway.

Effective amelioration of ischemia/reperfusion (I/R)-induced intestinal injury and revealing its mechanisms remain the challenges in both preclinic and clinic. Potential mechanisms of naringin in ameliorating I/R-induced intestinal injury remain unknown. Based on pre-experiments, I/R-injured rat intestine in vivo and hypoxia-reoxygenation (H/R)-injured IEC-6 cells in vitro were used to verify that naringin-alleviated I/R-induced intestinal injury was mediated via deactivating cGAS-STING signaling pathway. Naringin improved intestinal damage using hematoxylin and eosin staining and decreased alanine aminotransferase and aspartate aminotransferase contents in plasma. Naringin decreased inflammation characterized by reducing IL-6, IL-1ß, TNF-α, and IFN-ß contents in both plasma and IEC-6 cells. Naringin mitigated oxidative stress via recovering superoxide dismutase, glutathione, and malondialdehyde levels in the I/R-injured intestine. Naringin reduced the expression of apoptotic proteins, including Bax, caspase-3, and Bcl-2, and reduced terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling-positive cells both in vivo and in vitro, and decreased Hoechst 33342 signals in vitro. cGAS, STING, p-TBK1, p-IRF3, and NF-κB expressions were up-regulated both in vivo and in vitro respectively and the up-regulated indexes were reversed by naringin. Transfection of cGAS-siRNA and cGAS-cDNA significantly down-regulated and up-regulated cGAS-STING signaling-related protein expressions, respectively, and partially weakened naringin-induced amelioration on these indexes, suggesting that deactivation of cGAS-STING signaling is the crucial target for naringin-induced amelioration on I/R-injured intestine.

Interbacterial Chemical Communication-Triggered Nascent Proteomics.

Metabolic labeling with clickable noncanonical amino acids has enabled nascent proteome profiling, which can be performed in a cell-type-specific manner. However, nascent proteomics in an intercellular communication-dependent manner remains challenging. Here we develop communication-activated profiling of protein expression (CAPPEX), which integrates the LuxI/LuxR quorum sensing circuit with the cell-type-specific nascent proteomics method to enable selective click-labeling of newly synthesized proteins in a specific bacterium upon receiving chemical signals from another reporter bacterium. CAPPEX reveals that E. coli competes with Salmonella for tryptophan as the precursor for indole, and the resulting indole suppressed the expression of virulence factors in Salmonella. This tryptophan-indole axis confers attenuation of Salmonella invasion in host cells and living mice. The CAPPEX strategy should be widely applicable for investigating various interbacterial communication processes.

Bioorthogonal Lanthanide Molecular Probes for Near-Infrared Fluorescence and Mass Spectrometry Imaging.

We report here the development of clickable and highly near-infrared (NIR) fluorescent lanthanide (Ln) complexes for bioorthogonal labeling of biomolecules. These azide- or alkyne-functionalized Ln complexes are hydrophilic and fluorogenic, exhibiting a strong increase of NIR fluorescence upon conjugation with biomolecules. Metabolic labeling of biomolecules with azide or alkyne, followed by click labeling with the Ln complexes, enables NIR fluorescence (NIRF) imaging of DNA, RNA, proteins, and glycans in cells. Furthermore, multicolor imaging is performed by combining click-labeling with the Ln complexes and immunostaining. In addition, the Ln complexes is compatible with click-expansion microscopy (click-ExM), which enables high-resolution NIRF imaging of cellular glycoproteins. Finally, the Ln complexes can be used for time-of-flight secondary-ion mass spectrometry (ToF-SIMS) imaging, thus achieving the first example of dual-modal imaging combining NIRF and SIMS microscopies.

Effect of Al Content on Microstructure and Mechanical Property of Nanocomposite TiAlSiN Thin Films.

TiAlSiN thin films exhibit high hardness due to the multicomponent and multiphase structures. Effects of Si on TiAlSiN microstructure and mechanical properties have been well studied. However, there is insufficient information on the effects of Al. Thus, in this work, Al-contained nanocomposite nc-TiN/a-SiNx thin films with Al up to 10 at.% were prepared by magnetron co-sputtering Ti, Al and Si3N4 targets in Ar/N2 gas atmosphere. The effects of Al on TiAlSiN microstructure and mechanical property were well studied. Adjusting Al/(Ti+Al) target power ratio tuned chemical composition, microstructure and consequently mechanical properties. With increase of Al/(Ti+Al) target power ratio from 0 to 0.2, Al content increased from 0 to 10 at.%. X-ray photoelectron spectroscope results showed that aluminum nitride was formed. X-ray diffraction results confirmed that the nanocrystalline phase TiN was with Si and Al in its structure, or (Ti, Al, Si) N. The matrix was amorphous silicon nitride containing aluminum nitride. Nanoindentation hardness of the thin films remained at about 28 GPa till Al addition was increased to 3.1 at.%.

Mechanistic Investigation and Multiplexing of Liposome-Assisted Metabolic Glycan Labeling.

Metabolic labeling of glycans with bioorthogonal reporters has been widely used for glycan imaging and glycoproteomic profiling. One of the intrinsic limitations of metabolic glycan labeling is the lack of cell-type selectivity. The recently developed liposome-assisted bioorthogonal reporter (LABOR) strategy provides a promising means to overcome this limitation, but the mechanism of LABOR has not been investigated in detail. In this work, we performed a mechanistic study on LABOR and explored its multiplexing capability. Our studies support an endocytosis-salvage mechanism. The ligand-targeted liposomes encapsulating azidosugars are internalized into the endosome via the receptor-mediated endocytosis. Unlike the conventional drug delivery, LABOR does not rely on the endosomal escape pathways. Rather, the liposomes are allowed to enter the lysosome, inside which the azidosugars are released from the liposomes. The released azidosugars then intercept the salvage pathways of monosaccharides and get transported into the cytosol by lysosomal sugar transporters. Based on this mechanism, we expanded the scope of LABOR by evaluating a series of ligand-receptor pairs for targeting sialoglycans in various cell types. Different ligand types including small molecules, antibodies, aptamers, and peptides could be easily implemented into LABOR. Finally, we demonstrated that the sialoglycans in two distinct cell populations in a co-cultured system could be selectively labeled with two distinct chemical reporters by performing a multiplexed LABOR labeling.

Hybrid Indicators for Fast and Sensitive Voltage Imaging.

Membrane voltage is an important biophysical signal that underlies intercellular electrical communications. A fluorescent voltage indicator is presented that enables the investigation of electrical signaling at high spatial resolution. The method is built upon the site-specific modification of microbial rhodopsin proteins with organic fluorophores, resulting in a hybrid indicator scaffold that is one of the most sensitive and fastest orange-colored voltage indicators developed to date. We applied this technique to optically map electrical connectivity in cultured cells, which revealed gap junction-mediated long-range coupling that spanned over hundreds of micrometers.

Ultrasensitive biosensing platform based on the luminescence quenching ability of plasmonic palladium nanoparticles.

An ultrasensitive biosensing platform for DNA and protein detection is constructed based on the luminescence quenching ability of plasmonic palladium nanoparticles (PdNPs). By growing the particles into large sizes (ca. 30 nm), the plasmonic light absorption of PdNPs is broadened and extended to the visible range with extinction coefficients as high as 10(9)  L mol(-1) cm(-1) , enabling complete quenching of fluorescent dyes that emit at diverse ranges and that are tagged to bioprobes. Meanwhile the nonspecific quenching of the dyes (not bound to probes) is negligible, leading to extremely low background signal. Utilizing the affinity of PdNPs towards bioprobes, such as single-stranded (ss) DNA and polypeptide molecules, which is mainly assigned to the coordination interaction, nucleic acid assays with a quantification limit of 3 pM target DNA and protein assay are achieved with a simple mix-and-detect strategy based on the luminescence quenching-and-recovery protocol. This is the first demonstration of biosensing employing plasmonic absorption of nanopalladium, which offers pronounced sensing performances and can be reasonably expected for wide applications.

Study on Microstructure and Tribological Mechanism of Mo Incorporated (AlCrTiZr)N High-Entropy Ceramics Coatings Prepared by Magnetron Sputtering.

(AlCrTiZrMox)N coatings with varying Mo content were successfully prepared using a multi-target co-deposition magnetron sputtering system. The results reveal that the Mo content significantly affects the microstructure, hardness, fracture toughness, and tribological behavior of the coatings. As the Mo content in the coatings increases gradually, the preferred orientation changes from (200) to (111). The coatings consistently exhibit a distinct columnar structure. Additionally, the hardness of the coatings increases from 24.39 to 30.24 GPa, along with an increase in fracture toughness. The friction coefficient is reduced from 0.72 to 0.26, and the wear rate is reduced by 10 times. During the friction process, the inter-column regions of the coatings are initially damaged, causing the wear track to exhibit a wavy pattern. Greater frictional heat is generated at the crest of the wave, resulting in the formation of a MoO2 lubricating layer. The friction reaction helps to reduce the shear force during friction, demonstrating the lower friction coefficient of the (AlCrTiZrMox)N coatings. Both the hardness and fracture toughness work together to reduce the wear rate, and the (AlCrTiZrMox)N coatings show excellent wear resistance. Most notably, although the columnar structure plays a negative role in the hardness, it contributes greatly to the wear resistance.

MicroRNA-122a aggravates intestinal ischemia/reperfusion injury by promoting pyroptosis via targeting EGFR-NLRP3 signaling pathway.

Multiple studies have confirmed the significance of microRNA (miR)-122a in disease regulation. However, its impact on ischaemia/reperfusion (I/R) injury is unknown. In this study, we propose that the promoting role of miR-122a exists in I/R injuries. Two models, including hypoxia/reoxygenation (H/R)-injured IEC-6 cells in vitro and ischemia/reperfusion (I/R)-injured C57BL/6 mice intestinal tissues in vivo, were used to verify our purpose. Applying dual-luciferase reporter assays and transfection tests, the regulatory impacts of miR-122a were examined by promoting pyroptosis on intestinal I/R injury via targeting epidermal growth factor receptor (EGFR)-NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) signaling pathway. Both H/R-injured IEC-6 cells and I/R-injured mice intestinal tissues had elevated miR-122a expression, which targeted EGFR directly. Increased miR-122a expression significantly inhibited EGFR activity, decreased EGFR mRNA and protein expression, increased NLRP3 mRNA and protein expression, and up-regulated caspase 1, N-GSDMD, ASC, IL-1ß, and IL-18 protein expression to promote pyroptosis. Furthermore, in IEC-6 cells, a miR-122a inhibitor and an EGFR-overexpression plasmid significantly reduced pyroptosis and alleviated intestinal I/R injury via activating the EGFR-NLRP3 signaling pathway, showing that miR-122a is very essential for regulating intestinal I/R injury. In brief, miR-122a promotes pyroptosis by inhibiting the EGFR-NLRP3 signaling pathway, which should be evaluated as a therapeutic target for intestinal I/R injury.

Sparse deconvolution improves the resolution of live-cell super-resolution fluorescence microscopy.

A main determinant of the spatial resolution of live-cell super-resolution (SR) microscopes is the maximum photon flux that can be collected. To further increase the effective resolution for a given photon flux, we take advantage of a priori knowledge about the sparsity and continuity of biological structures to develop a deconvolution algorithm that increases the resolution of SR microscopes nearly twofold. Our method, sparse structured illumination microscopy (Sparse-SIM), achieves ~60-nm resolution at a frame rate of up to 564 Hz, allowing it to resolve intricate structures, including small vesicular fusion pores, ring-shaped nuclear pores formed by nucleoporins and relative movements of inner and outer mitochondrial membranes in live cells. Sparse deconvolution can also be used to increase the three-dimensional resolution of spinning-disc confocal-based SIM, even at low signal-to-noise ratios, which allows four-color, three-dimensional live-cell SR imaging at ~90-nm resolution. Overall, sparse deconvolution will be useful to increase the spatiotemporal resolution of live-cell fluorescence microscopy.

Effect of fluorine incorporation on long-term stability of magnesium-containing hydroxyapatite coatings.

Dissolution resistance and adhesion strength are two main concerns for long-term stability of surface coated metal implants. In this study, fluorine ions are incorporated into magnesium-containing hydroxyapatite coatings (MgF(y)HA) via sol-gel method to improve the long-term stability of the implants. Surface and interface are studied in terms of phases, depth profiling and chemical bonds by grazing incidence X-ray diffraction, glow discharge optical emission spectroscopy and X-ray photoelectron spectroscopy. The long-term stability is evaluated by dissolution and pull-off test. The results show that fluorine promotes the incorporation of magnesium in HA lattice. The elemental interdiffusion and chemical bonding take place at the coating/substrate interface. Both the dissolution resistance and the adhesion strength are enhanced by fluorine incorporation, thus the long-term stability of the implant is improved.

catena-Poly[[diaqua-bis-(formato-κO)cobalt(II)]-µ(2)-2,6-bis-(pyridin-4-yl)-4,4'-bipyridine-κN:N].

In the title complex, [Co(CHO(2))(2)(C(20)H(14)N(4))(H(2)O)(2)](n), the Co(II) ion, lying on an inversion center, is six-coordinated by two O atoms from two monodentate formate ligands, two N atoms from two 2,6-bis-(pyridin-4-yl)-4,4'-bipyridine (4-pybpy) ligands and two water mol-ecules, displaying an octa-hedral geometry. The 4-pybpy ligand, having a twofold rotation axis, functions in a bridging coordination mode, connecting the Co(II) ions into a corrugated chain along [[Formula: see text]01]. The chains are further linked into a three-dimensional supra-molecular network by O-H⋯O, C-H⋯N and C-H⋯O hydrogen bonds and π-π stacking inter-actions between the pyridine rings [centroid-to-centroid distance = 3.743 (2) Å].

Quantitative and Site-Specific Chemoproteomic Profiling of Protein O-GlcNAcylation in the Cell Cycle.

Mammalian cell cycle is a central process for tissue growth and maintenance. Protein O-linked ß-N-acetylglucosamine (O-GlcNAc) modification has been found to occur on several important cell cycle regulators. However, the O-GlcNAcylated proteome has not been extensively profiled during cell cycle progression. Herein, we report a quantitative profiling of protein O-GlcNAcylation sites in cell proliferation, by using an O-GlcNAc chemoproteomic strategy. In HeLa cells, a total of 902, 439, and 872 high-confidence O-GlcNAcylation sites distributed on 414, 265, and 425 proteins are identified in the interphase, early mitosis, and mitotic exit stages, respectively. The identified O-GlcNAcylation events occur on a variety of important regulators, which are involved in the processes of cell division, DNA repair, and cell death. Furthermore, we show that O-GlcNAcylation is dynamically regulated in a cell cycle stage-dependent manner. Our results provide a valuable resource for investigating the functional roles of O-GlcNAc in the mammalian cell cycle.

Next-generation unnatural monosaccharides reveal that ESRRB O-GlcNAcylation regulates pluripotency of mouse embryonic stem cells.

Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per-O-acetylated, which, however, can induce a long-overlooked side reaction, non-enzymatic S-glycosylation. Herein, we develop 1,3-di-esterified N-azidoacetylgalactosamine (GalNAz) as next-generation chemical reporters for metabolic glycan labeling. Both 1,3-di-O-acetylated GalNAz (1,3-Ac2GalNAz) and 1,3-di-O-propionylated GalNAz (1,3-Pr2GalNAz) exhibit high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr2GalNAz in mouse embryonic stem cells (mESCs), we identify ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We show that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase at serine 25, which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG.

Fluorescence resonance energy transfer biosensor between upconverting nanoparticles and palladium nanoparticles for ultrasensitive CEA detection.

An ultrasensitive biosensor for carcinoembryonic antigen (CEA) was constructed based on fluorescence resonance energy transfer (FRET) between upconverting nanoparticles (UCPs) and palladium nanoparticles (PdNPs). PdNPs was synthesized by the addition of a solution of Na2PdCl4 into a mixture of N2H4·H2O as the reducing agent and 11-mercaptoundecanoic acid (MUDA) as the stabilizer. The CEA aptamer (5'-NH2-ATACCAGCTTATTCAATT-3') was conjugated to hexanedioic acid (HDA) modified UCPs (HDA-UCPs) through an EDC-NHS coupling protocol. The coordination interaction between nitrogen functional groups of the CEA aptamer and PdNPs brought UCPs and PdNPs in close proximity, which resulted in the fluorescence quenching of UCPs to an extent of 85%. And the non-specific fluorescence quenching caused by PdNPs towards HDA-UCPs was negligible. After the introduction of CEA into the UCPs-CEA aptamer-PdNPs fluorescence quenching system, the CEA aptamer preferentially combined with CEA accompanied by the conformational change which weakened the coordination interaction between the CEA aptamer and PdNPs. So fluorescence recovery of UCPs was observed and a linear relationship between the fluorescence recovery of UCPs and the concentration of CEA was obtained in the range from 2pg/mL to 100pg/mL in the aqueous buffer with the detection limit of 0.8pg/mL. The ultrasensitive detection of CEA was also realized in diluted human serum with a linear range from 4pg/mL to 100pg/mL and a detection limit of 1.7pg/mL. This biosensor makes the most of the high quenching ability of PdNPs towards UCPs with negligible non-specific fluorescence quenching and has broad application prospects in biochemistry.