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.

Identifying the origin of local flexibility in a carbohydrate polymer.

Correlating the structures and properties of a polymer to its monomer sequence is key to understanding how its higher hierarchy structures are formed and how its macroscopic material properties emerge. Carbohydrate polymers, such as cellulose and chitin, are the most abundant materials found in nature whose structures and properties have been characterized only at the submicrometer level. Here, by imaging single-cellulose chains at the nanoscale, we determine the structure and local flexibility of cellulose as a function of its sequence (primary structure) and conformation (secondary structure). Changing the primary structure by chemical substitutions and geometrical variations in the secondary structure allow the chain flexibility to be engineered at the single-linkage level. Tuning local flexibility opens opportunities for the bottom-up design of carbohydrate materials.

Transcriptome Analysis Reveals Coexpression Networks and Hub Genes Involved in Papillae Development in Lilium auratum.

Lilium is a genus of important ornamental plants with many colouring pattern variations. Lilium auratum is the parent of Oriental hybrid lilies. A typical feature of L. auratum is the presence of red-orange special raised spots named papillae on the interior tepals. Unlike the usual raised spots, the papillae are slightly rounded or connected into sheets and usually have hairy tips. To elucidate the potential genes regulating papillae development in L. auratum, we performed high-throughput sequencing of its tepals at different stages. Genes involved in the flavonoid biosynthesis pathway were significantly enriched during the colouration of the papillae, and CHS, F3H, F3'H, FLS, DFR, ANS, and UFGT were significantly upregulated. To identify the key genes involved in the papillae development of L. auratum, we performed weighted gene coexpression network analysis (WGCNA) and further analysed four modules. In total, 51, 24, 1, and 6 hub genes were identified in four WGCNA modules, MEbrown, MEyellow, MEpurple, and MEred, respectively. Then, the coexpression networks were constructed, and important genes involved in trichome development and coexpressed with anthocyanin biosynthesis genes, such as TT8, TTG1, and GEM, were identified. These results indicated that the papillae are essentially trichomes that accumulate anthocyanins. Finally, we randomly selected 12 hub genes for qRT-PCR analysis to verify the accuracy of our RNA-Seq analysis. Our results provide new insights into the papillae development in L. auratum flowers.

Chemical Synthesis of the Highly Sterically Hindered Core Undecasaccharide of Helicobacter pylori Lipopolysaccharide for Antigenicity Evaluation with Human Serum.

Helicobacter pylori, listed as a human carcinogen by the Department of Health and Human Services, colonizes the gastric mucosa of more than half of the world's population. The individuals infected with H. pylori have a high risk to develop chronic gastritis, peptic ulcers, and even gastric cancer. The conserved core structure of H. pylori lipopolysaccharide (LPS) has been regarded as a promising candidate structure for development of a glycoconjugate vaccine targeting multiple serotypes. Here, we report a total synthesis of the core undecasaccharide of H. pylori LPS and its subunit antigens. The match and mismatch between the glycosyl donor and acceptor caused by the inert hydroxyl groups were addressed by a judicious choice of orthogonal protection strategies and glycosylation conditions. A combination of acyl remote participation and solvent effects has been applied for selective formation of the five 1,2-cis-glucosidic bonds. The high steric hindrance induced by the high carbon sugars and trinacriform architecture required that the core undecasaccharide was synthesized through a finely tuned linear assembly [2 + (1 + (3 + (1 + (1 + 3))))] rather than convergent strategies. An antigenicity evaluation using glycan microarrays showed that an α-(1 → 6)-glucan trisaccharide is recognized by IgG antibodies in sera of H. pylori-infected patients. The phosphate group of the inner core trisaccharide key epitope is very important for IgG recognition. These findings are an important step toward designing carbohydrate-based vaccines against H. pylori.

Compact substrate-removed thin-film lithium niobate electro-optic modulator featuring polarization-insensitive operation.

A compact polarization-insensitive electro-optic (EO) modulator, which allows the laser and modulator to be located at different locations while using a standard single-mode fiber to interconnect them, is highly desirable for 5G or future 6G wireless networks. Herein, we propose a modulator based on substrate-removed thin-film lithium niobate. The proposed device exhibits a polarization-dependent loss of 0.35 dB and on-chip loss of approximately 2 dB. The polarization insensitivity of the proposed device was experimentally demonstrated using a four-level pulse-amplitude modulation format at 50 Gbaud (100 Gb/ s).

Automated Assembly of Starch and Glycogen Polysaccharides.

Polysaccharides are Nature's most abundant biomaterials essential for plant cell wall construction and energy storage. Seemingly minor structural differences result in entirely different functions: cellulose, a ß (1-4) linked glucose polymer, forms fibrils that can support large trees, while amylose, an α (1-4) linked glucose polymer forms soft hollow fibers used for energy storage. A detailed understanding of polysaccharide structures requires pure materials that cannot be isolated from natural sources. Automated Glycan Assembly provides quick access to trans-linked glycans analogues of cellulose, but the stereoselective installation of multiple cis-glycosidic linkages present in amylose has not been possible to date. Here, we identify thioglycoside building blocks with different protecting group patterns that, in concert with temperature and solvent control, achieve excellent stereoselectivity during the synthesis of linear and branched α-glucan polymers with up to 20 cis-glycosidic linkages. The molecules prepared with the new method will serve as probes to understand the biosynthesis and the structure of α-glucans.

Low-loss edge-coupling thin-film lithium niobate modulator with an efficient phase shifter: publisher's note.

This publisher's note contains corrections to Opt. Lett.46, 1478 (2021)OPLEDP0146-959210.1364/OL.418996.

Low-loss edge-coupling thin-film lithium niobate modulator with an efficient phase shifter.

Thin-film lithium-niobate-on-insulator (LNOI) is a very attractive platform for optical interconnect and nonlinear optics. It is essential to enable lithium niobate photonic integrated circuits with low power consumption. Here we present an edge-coupling Mach-Zehnder modulator on the platform with low fiber-chip coupling loss of 0.5 dB/facet, half-wave voltage Vπ of 2.36 V, electro-optic (EO) bandwidth of 60 GHz and an efficient thermal-optic phase shifter with half-wave power of 6.24 mW. In addition, we experimentally demonstrate single-lane 200 Gbit/s data transmission utilizing a discrete multi-tone signal. The LNOI modulator demonstrated here shows great potential in energy-efficient large-capacity optical interconnects.

Targeted Chemical Modifications Identify Key Features of Carbohydrate Assemblies and Generate Tailored Carbohydrate Materials.

The molecular level description of carbohydrate assemblies is hampered by their structural complexity and the lack of suitable analytical methods. Here, we employed systematic chemical modifications to identify key non-covalent interactions that triggered the supramolecular assembly of a disaccharide model. While some modifications disrupted the supramolecular organization, others were tolerated, delivering important information on the aggregation process. The screening identified new geometries, including nanotubes, and twisted ribbons that were characterized with electron tomography and electron diffraction (ED) methods. This work demonstrates that the combination of synthetic chemistry and ED methods is a powerful tool to draw correlations between the molecular structure and the nanoscale architecture of carbohydrate assemblies.

Legionella effector SetA as a general O-glucosyltransferase for eukaryotic proteins.

The identification of host protein substrates is key to understanding effector glycosyltransferases secreted by pathogenic bacteria and to using them for glycoprotein engineering. Here we report a chemical method for tagging, enrichment, and site-specific proteomic profiling of effector-modified proteins in host cells. Using this method, we discover that Legionella effector SetA α-O-glucosylates various eukaryotic proteins by recognizing a S/T-X-L-P/G sequence motif, which can be exploited to site-specifically introduce O-glucose on recombinant proteins.

Exploring the Molecular Conformation Space by Soft Molecule-Surface Collision.

Biomolecules function by adopting multiple conformations. Such dynamics are governed by the conformation landscape whose study requires characterization of the ground and excited conformation states. Here, the conformational landscape of a molecule is sampled by exciting an initial gas-phase molecular conformer into diverse conformation states, using soft molecule-surface collision (0.5-5.0 eV). The resulting ground and excited molecular conformations, adsorbed on the surface, are imaged at the single-molecule level. This technique permits the exploration of oligosaccharide conformations, until now, limited by the high flexibility of oligosaccharides and ensemble-averaged analytical methods. As a model for cellulose, cellohexaose chains are observed in two conformational extremes, the typical "extended" chain and the atypical "coiled" chain-the latter identified as the gas-phase conformer preserved on the surface. Observing conformations between these two extremes reveals the physical properties of cellohexaose, behaving as a rigid ribbon that becomes flexible when twisted. The conformation space of any molecule that can be electrosprayed can now be explored.

Compact high-efficiency four-mode vortex beam generator within the telecom C-band.

Vortex beams carrying orbital angular momentum have attracted a great deal of attention over the past few years. An integrated vortex beam generator with high efficiency is desirable for wide-ranging applications. Here we demonstrate a highly efficient silicon photonic integrated vortex beam generator based on superposed holographic fork gratings. A metal mirror is used to enhance emission efficiency by reflecting the power leaking down to the substrate back to air. Experimental characterization confirms that the emission efficiency of the generator increases by ${\sim} 5\,{\rm dB}$∼5dB. Moreover, the present device shows preferable features of broadband, polarization diversity, and compact footprint.

Compact high-efficiency four-mode vortex beam generator within the telecom C-band: publisher's note.

This publisher's note contains corrections to Opt. Lett.45, 1607 (2020)OPLEDP0146-959210.1364/OL.385878.

Automated access to well-defined ionic oligosaccharides.

Ionic polysaccharides are part of many biological events, but lack structural characterisation due to challenging purifications and complex synthesis. Four monosaccharides bearing modifications not found in nature are used for the automated synthesis of a collection of ionic oligosaccharides. Structural analysis reveals how the charge pattern affects glycan conformation.

Quantitative time-resolved chemoproteomics reveals that stable O-GlcNAc regulates box C/D snoRNP biogenesis.

O-linked GlcNAcylation (O-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of O-GlcNAcylated proteins, we developed a quantitative time-resolved O-linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an O-GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533 O-GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of O-GlcNAc or degradation of protein backbones. The stability of those hyperstable O-GlcNAcylated proteins was more sensitive to O-GlcNAcylation inhibition compared with the more dynamic populations. Among the hyperstable population were three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs): fibrillarin (FBL), nucleolar protein 5A (NOP56), and nucleolar protein 5 (NOP58). We showed that O-GlcNAcylation stabilized these proteins and was essential for snoRNP assembly. Blocking O-GlcNAcylation on FBL altered the 2'-O-methylation of rRNAs and impaired cancer cell proliferation and tumor formation in vivo.

Genome-Wide Analysis of the Cyclin Gene Family and Their Expression Profile in Medicago truncatula.

Cyclins, together with highly conserved cyclin-dependent kinases (CDKs), play an important role in the process of cell cycle in plants, but less is known about the functions of cyclins in legume plants, especially Medicago truncatula. Our genome-wide analysis identified 58, 103, and 51 cyclin members in the M. truncatula, Glycine max, and Phaseolus vulgaris genomes. Phylogenetic analysis suggested that these cyclins could be classified into 10 types, and the CycB-like types (CycBL1-BL8) were the specific subgroups in M. truncatula, which was one reason for the expansion of the B-type in M. truncatula. All putative cyclin genes were mapped onto their own chromosomes of each genome, and 9 segmental duplication gene pairs involving 20 genes were identified in M. truncatula cyclins. Determined by quantitative real-time PCR, the expression profiling suggested that 57 cyclins in M. truncatula were differentially expressed in 9 different tissues, while a few genes were expressed in some specific tissues. Using the publicly available RNAseq data, the expression of Mtcyclins in the wild-type strain A17 and three nodule mutants during rhizobial infection showed that 23 cyclins were highly upregulated in the nodulation (Nod) factor-hypersensitive mutant sickle (skl) mutant after 12 h of rhizobium inoculation. Among these cyclins, six cyclin genes were also specifically expressed in roots and nodules, which might play specific roles in the various phases of Nod factor-mediated cell cycle activation and nodule development. Our results provide information about the cyclin gene family in legume plants, serving as a guide for further functional research on plant cyclins.

Traceless Photolabile Linker Expedites the Chemical Synthesis of Complex Oligosaccharides by Automated Glycan Assembly.

Automated glycan assembly (AGA) aims at accelerating access to synthetic oligosaccharides to meet the demand for defined glycans as tools for molecular glycobiology. The linkers used to connect the growing glycan chain to the solid support play a pivotal role in the synthesis strategy as they determine all chemical conditions used during the synthesis and the form of the glycan obtained at the end of it. Here, we describe a traceless photolabile linker used to prepare carbohydrates with a free reducing end. Modification of the o-nitrobenzyl scaffold of the linker is key to high yields and compatibility with the AGA workflow. The assembly of an asymmetrical biantennary N-glycan from oligosaccharide fragments prepared by AGA and linear as well as branched ß-oligoglucans is described to illustrate the power of the method. These substrates will serve as standards and biomarkers to examine the unique specificity of glycosyl hydrolases.

CCR4 Expression Is Associated With Poor Prognosis in Patients With Early Stage (pN0) Oral Tongue Cancer.

PURPOSE: Chemokine receptors are involved in tumor metastasis and can predict poor prognosis; however, the expression and clinicopathologic relevance of chemokine receptors in early-stage cancer remain largely unknown. This study measured the association between chemokine (C-C motif) receptor-4 (CCR4) expression and prognosis in patients with histologically node-negative (pN0) oral tongue cancer. MATERIALS AND METHODS: A retrospective analysis of CCR4 expression data from a consecutive case series of patients with pN0 oral cancer tongue was conducted. The expression of CCR4 by immunohistochemistry was investigated and the association between CCR4 expression and clinicopathologic variables and overall and disease-free survivals was evaluated using Kaplan-Meier analysis and a Cox regression model. RESULTS: CCR4 expression was examined in 128 human tongue cancerous samples (109 tongue squamous cell carcinomas [TSCCs] and 19 other types) and 10 normal tongue samples and was found to be highly expressed in tumor tissues compared with normal tissues. CCR4 expression was observed in 64.2% of patients with TSCC and showed a significant association with tumor stage (P = .037). Patients with CCR4-positive expression exhibited poorer overall and disease-free survivals compared with those with CCR4-negative expression (P < .001 and P = .001), and CCR4-positive expression was an independent factor of unfavorable overall and disease-free survivals (P = .002 and P = .007). CONCLUSIONS: This study identified CCR4 as a potential prognostic biomarker for recurrence and survival of patients with pN0 oral tongue cancer. Thus, CCR4 might be a possible therapeutic target for patients with early-stage cancer.

In vivo metabolic labeling of sialoglycans in the mouse brain by using a liposome-assisted bioorthogonal reporter strategy.

Mammalian brains are highly enriched with sialoglycans, which have been implicated in brain development and disease progression. However, in vivo labeling and visualization of sialoglycans in the mouse brain remain a challenge because of the blood-brain barrier. Here we introduce a liposome-assisted bioorthogonal reporter (LABOR) strategy for shuttling 9-azido sialic acid (9AzSia), a sialic acid reporter, into the brain to metabolically label sialoglycoconjugates, including sialylated glycoproteins and glycolipids. Subsequent bioorthogonal conjugation of the incorporated 9AzSia with fluorescent probes via click chemistry enabled fluorescence imaging of brain sialoglycans in living animals and in brain sections. Newly synthesized sialoglycans were found to widely distribute on neuronal cell surfaces, in particular at synaptic sites. Furthermore, large-scale proteomic profiling identified 140 brain sialylated glycoproteins, including a wealth of synapse-associated proteins. Finally, by performing a pulse-chase experiment, we showed that dynamic sialylation is spatially regulated, and that turnover of sialoglycans in the hippocampus is significantly slower than that in other brain regions. The LABOR strategy provides a means to directly visualize and monitor the sialoglycan biosynthesis in the mouse brain and will facilitate elucidating the functional role of brain sialylation.

Systematic Hydrogen-Bond Manipulations To Establish Polysaccharide Structure-Property Correlations.

A dense hydrogen-bond network is responsible for the mechanical and structural properties of polysaccharides. Random derivatization alters the properties of the bulk material by disrupting the hydrogen bonds, but obstructs detailed structure-function correlations. We have prepared well-defined unnatural oligosaccharides including methylated, deoxygenated, deoxyfluorinated, as well as carboxymethylated cellulose and chitin analogues with full control over the degree and pattern of substitution. Molecular dynamics simulations and crystallographic analysis show how distinct hydrogen-bond modifications drastically affect the solubility, aggregation behavior, and crystallinity of carbohydrate materials. This systematic approach to establishing detailed structure-property correlations will guide the synthesis of novel, tailor-made carbohydrate materials.