Efficient Enzymatic Glycan Engineering of Extracellular Vesicles Using Nanomaterial-Interfaced Microfluidics.

Extracellular vesicles (EVs) present a promising modality for numerous biological and medical applications, including therapeutics. Developing facile methods to engineer EVs is essential to meeting the rapidly expanding demand for various functionalized EVs in these applications. Herein, we developed a technology that integrates enzymatic glycoengineering and microfluidics for effective EV functionalization.

Preparation of recombinant neuritin protein.

Neuritin plays an important role in promoting nerve injury repair and maintaining synaptic plasticity, making it a potential therapeutic target for the treatment of nerve injury and neurodegenerative diseases. The present study aimed to obtain an active, unlabeled neuritin protein. Initially, a neuritin protein expression system with an enterokinase site was constructed in Escherichia coli.

Sensitive Method To Analyze Cell Surface GPI-Anchored Proteins Using DNA Hybridization Chain Reaction-Mediated Signal Amplification.

GPI-anchored proteins (GPI-APs) are ubiquitous and essential but exist in low abundances on the cell surface, making their analysis and investigation especially challenging. To tackle the problem, a new method to detect and study GPI-APs based upon GPI metabolic engineering and DNA-facilitated fluorescence signal amplification was developed. In this context, cell surface GPI-APs were metabolically engineered using azido-inositol derivatives to introduce an azido group.

Monophosphoryl Lipid A-Rhamnose Conjugates as a New Class of Vaccine Adjuvants.

Adjuvant is an integral part of all vaccine formulations but only a few adjuvants with limited efficacies or application scopes are available. Thus, developing more robust and diverse adjuvants is necessary. To this end, a new class of adjuvants having α- and β-rhamnose (Rha) attached to the 1- and 6'-positions of monophosphoryl lipid A (MPLA) was designed, synthesized, and immunologically evaluated in mice.

Metabolic-Glycoengineering-Enabled Molecularly Specific Acoustic Tweezing Cytometry for Targeted Mechanical Stimulation of Cell Surface Sialoglycans.

In this study, we developed a novel type of dibenzocyclooctyne (DBCO)-functionalized microbubbles (MBs) and validated their attachment to azide-labelled sialoglycans on human pluripotent stem cells (hPSCs) generated by metabolic glycoengineering (MGE). This enabled the application of mechanical forces to sialoglycans on hPSCs through molecularly specific acoustic tweezing cytometry (mATC), that is, displacing sialoglycan-anchored MBs using ultrasound (US). It was shown that subjected to the acoustic radiation forces of US pulses, sialoglycan-anchored MBs exhibited significantly larger displacements and faster, more complete recovery after each pulse than integrin-anchored MBs, indicating that sialoglycans are more stretchable and elastic than integrins on hPSCs in response to mechanical force.

Spin-labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells.

As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction.

A Biotinylated Glycosylphosphatidylinositol (GPI) as the Universal Platform To Access GPI-Anchored Protein Analogues.

A glycosylphosphatidylinositol (GPI) derivative with biotin linked to its mannose III 6-O-position was prepared by a convergent strategy. This biotinylated GPI was demonstrated to bind avidinated proteins readily through biotin-avidin interaction and, therefore, can serve as a universal platform to access various biologically significant GPI-anchored protein analogues.

Investigation of Glycosylphosphatidylinositol (GPI)-Plasma Membrane Interaction in Live Cells and the Influence of GPI Glycan Structure on the Interaction.

Glycosylphosphatidylinositols (GPIs) need to interact with other components in the cell membrane to transduce transmembrane signals. A bifunctional GPI probe was employed for photoaffinity-based proximity labelling and identification of GPI-interacting proteins in the cell membrane. This probe contained the entire core structure of GPIs and was functionalized with photoreactive diazirine and clickable alkyne to facilitate its crosslinking with proteins and attachment of an affinity tag.

Spin-labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells.

As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction.

Different Biophysical Properties of Cell Surface α2,3- and α2,6-Sialoglycans Revealed by Electron Paramagnetic Resonance Spectroscopic Studies.

Sialoglycans on HeLa cells were labeled with a nitroxide spin radical through enzymatic glycoengineering (EGE)-mediated installation of azide-modified sialic acid (Neu5Ac9N) and then click reaction-based attachment of a nitroxide spin radical. α2,6-Sialyltransferase (ST) Pd2,6ST and α2,3-ST CSTII were used for EGE to install α2,6- and α2,3-linked Neu5Ac9N, respectively. The spin-labeled cells were analyzed by X-band continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy to gain insights into the dynamics and organizations of cell surface α2,6- and α2,3-sialoglycans.

Labeling cell surface glycosylphosphatidylinositol-anchored proteins through metabolic engineering using an azide-modified phosphatidylinositol.

Glycosylphosphatidylinositol (GPI) anchorage is one of the most common mechanisms to attach proteins to the plasma membrane of eukaryotic cells. GPI-anchored proteins (GPI-APs) play a critical role in many biological processes but are difficult to study. Here, a new method was developed for the effective and selective metabolic engineering and labeling of cell surface GPI-APs with an azide-modified phosphatidylinositol (PI) as the biosynthetic precursor of GPIs.

Enzymatic glycoengineering-based spin labelling of cell surface sialoglycans to enable their analysis by electron paramagnetic resonance (EPR) spectroscopy.

A novel method for spin labelling of sialoglycans on the cell surface is described. C9-Azido sialic acid was linked to glycans on live cells CSTII-catalysed α2,3-sialylation utilizing azido-sialic acid nucleotide as a sialyl donor, which was followed by attachment of a spin label to the azide click reaction. It enables the study of cell surface sialoglycans by EPR spectroscopy.

Direct access to various C3-substituted sialyl glycal derivatives from 3-iodo-sialyl glycals.

A new and efficient method was developed for the synthesis of C3-substituted sialyl glycals that are useful for novel sialidase inhibitor discovery. This method was based on the cross-coupling reactions of 3-iodo-sialyl glycal methyl ester with boronic acids, alkenes and alkynes to directly introduce various functional groups to the sialyl glycal C3-position. A series of C3-aryl, alkyl, alkenyl, and alkynyl derivatives of sialyl glycal were efficiently and conveniently synthesized for the first time by this method, which has demonstrated its wide application scope.

Synthesis of a dimer of the repeating unit of type Ia Group B extracellular capsular polysaccharide and immunological evaluations of related protein conjugates.

Type Ia group B (GBS) is one of the major causes of fatal infections in neonates. Its extracellular capsular polysaccharide (CPS) is a useful target for the development of anti-type Ia GBS vaccines. To explore the structure-activity relationships of type Ia GBS CPS and design more effective vaccines, a dimer of the branched pentasaccharide repeating unit of this CPS was synthesized by a highly convergent strategy highlighted by constructing the key intermediate via one-pot iterative glycosylation and imposing two side chains in one step via dual glycosylation.

Comparative immunological studies of tumor-associated Lewis X, Lewis Y, and KH-1 antigens.

Tumor-associated carbohydrate antigens Lewis X (Le), Lewis Y (Le), and KH-1 are useful targets for cancer immunotherapy. In this regard, an insight into the structure-immunogenicity relationships of these antigens is important but this has not been systematically investigated yet. In the current study, Le, Le, and KH-1 antigens with a lactose unit at the reducing end as a spacer were synthesized and coupled with keyhole limpet hemocyanin (KLH) protein.

An investigation of the reactions between azido alcohols and phosphoramidites.

The reactions of several β-, γ-, and δ-azido alcohols with dibenzyl and dimethyl -diisopropylphosphoramidites were examined. Detailed analysis of the intermediates and products formed from the reactions under different conditions provided useful information to gain insights into their mechanisms involving intramolecular Staudinger reaction, as well as the structure-reactivity relationships of both substrates. The reactions of γ- and δ-azido alcohols with dibenzyl -diisopropylphosphoramidite could produce 6- and 7-membered cyclic phosphoramidates, thereby providing a new synthetic method for these biologically important molecules.

Synthesis of Lewis Y Analogues and Their Protein Conjugates for Structure-Immunogenicity Relationship Studies of Lewis Y Antigen.

Analogues of cancer-associated Lewis Y (Le) antigen with varying structures at the reducing end were synthesized by a highly efficient strategy involving one-pot preactivation-based iterative glycosylation to obtain the key tetra-/pentasaccharide intermediates, which was followed by stereoselective fucosylation. After global deprotection, these oligosaccharides were coupled with carrier protein keyhole limpet hemocyanin. The resultant glycan-protein conjugates were subjected to immunological studies in mice.

Synthesis and Evaluation of GM2-Monophosphoryl Lipid A Conjugate as a Fully Synthetic Self-Adjuvant Cancer Vaccine.

An efficient method was developed for the synthesis of a GM2 derivative suitable for the conjugation with various biomolecules. This GM2 derivative was covalently linked to keyhole limpet hemocyanin (KLH) and monophosphoryl lipid A (MPLA) to form novel therapeutic cancer vaccines. Immunological evaluations of the resultant conjugates in mice revealed that they elicited robust GM2-specific overall and IgG antibody responses.

Synthesis of biotin-labelled core glycans of GPI anchors and their application in the study of GPI interaction with pore-forming bacterial toxins.

A convergent strategy was developed for the first-time synthesis of biotin-labeled GPI core glycans. These GPI conjugates are useful for various biological studies showcased by their application in the scrutiny of pore-forming bacterial toxin-GPI interaction, revealing that the phosphate group at the GPI inositol 1-O-position had a significant impact on GPI-toxin binding.

Biochemical studies of inositol N-acetylglucosaminyltransferase involved in mycothiol biosynthesis in Corynebacterium diphtheria.

Mycothiol (MSH) is the predominant low molecular weight thiol produced by actinomycetes, and it plays a pivotal role in the bacterial detoxication process. 1L-myo-Inositol-1-phosphate (1L-Ins-1-P) α-N-acetylglucosaminyltransferase (GlcNAc-T), known as MshA, is the only glycosyltransferase involved in MSH biosynthesis. In this work, the MshA from Corynebacterium diphtheria, named as CdMshA, was expressed, purified, and studied in detail.

Sulphation pattern analysis of chemically sulphated polysaccharide LbGp1 from Lycium barbarum by GC-MS.

The polysaccharide LbGp1 from Lycium barbarum L. was sulphated with sulphur trioxide-pyridine complex in DMF, yielding two sulphated polysaccharides, which were LbGp1-OL-SL with 13.7% sulphate content, and LbGp1-OL-SH with 27.

[Analysis of disyllabic mandarin speech test results of normal hearing persons with different ages].

Objective: To valuate test results of normal hearing persons with different ages using disyllabic mandarin speech test materials (MSTMs). Obtaining speech recognition threshold (SRT) and P-I function of different ages as clinical reference of hearing recovery and individual's ability to perceive and process speech.

Method: One hundred and twenty subjects with normal hearing who speak mandarin well in their daily lives were enrolled in this study and divided into four groups (18-30, 31-40, 41-50 and 51-60 years old).