High-throughput multiplex assays with mouse macrophages on pillar plate platforms.

It is challenging to rapidly identify immune responses that reflect the state and capability of immune cells due to complex heterogeneity of immune cells and their plasticity to pathogens and modulating molecules. Thus, high-throughput and easy-to-use cell culture and analysis platforms are highly desired for characterizing complex immune responses and elucidating their underlying mechanisms as well. In response to this need, we have developed a micropillar chip and a 384-pillar plate, printed mouse macrophage, RAW 264.7 cell line in alginate on the pillar plate platforms, and established multiplex cell-based assays to rapidly measure cell viability, expression of cell surface markers, and secretion of cytokines upon stimulation with model compound, lipopolysaccharide (LPS), as well as synthetic N-glycan polymers that mimic native glycoconjugates and could bind to lectin receptors on RAW 264.7 cells. Interestingly, changes in RAW 264.7 cell viability, expression levels of cell surface makers, and release of cytokines measured from the pillar plate platforms in the presence and absence of LPS were well correlated with those obtained from their counterpart, the 96-well plate with 2D-cultured macrophages. With this approach, we identified that α2,3-linked N-sialyllactose polymer has significant macrophage modulation activity among the N-glycan polymers tested. Therefore, we successfully demonstrated that our pillar plate platforms with 3D-cultured macrophages can streamline immune cell imaging and analysis in high throughput in response to compound stimulation. We envision that the pillar plate platforms could potentially be used for rapid characterization of immune cell responses and for screening immune cell-modulating molecules.

Synthesis and Evaluation of Protein-Phenylboronic Acid Conjugates as Lectin Mimetics.

Glycan-binding molecules, such as lectins, are very important tools for characterizing, imaging, or targeting glycans and are often involved in either physiological or pathological processes. However, their availability is far less compared to the diversity of native glycans. Therefore, development of lectin mimetics with desired specificity and affinity is in high demand. Boronic acid reacts with 1,2- and 1,3-diols of saccharides in aqueous media through reversible boronate ester formation and are regarded as synthetic lectin mimetics. In this study, bovine serum albumin (BSA)-phenylboronic acid (PBA) conjugates were synthesized in a density-controlled manner by targeting both aspartic and glutamic acids to afford lectin mimetics with multivalent PBA, as multivalency is a key factor for glycan recognition in both specificity and affinity. The resultant BSA-PBA conjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Their macrophage cell surface glycan-binding capacity was characterized by a competitive lectin-binding assay examined by flow cytometry, and 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed biocompatibility. These novel lectin mimetics will find a broad range of applications as they can be wittingly modified, altering binding specificity and capacity.

Recent Chemical Biology Approaches for Profiling Cell Surface Sialylation Status.

Sialic acids (SAs) often exist as the terminal sugars of glycans of either glycoproteins or glycolipids on the cell surface and thus are directly involved in biological processes, such as cell-cell, cell-ligand, and cell-pathogen interactions. Cell surface SA expression levels and their linkages are collectively termed cell surface sialylation status, which represent varying cellular states and contribute to the overall functionality of a cell. Accordingly, systemic and specific profiling of the cell surface sialyation status is critical in deciphering the structures and functions of cell surface glycoconjugates and the molecular mechanisms of their underlying biological processes. In recent decades, several advanced chemical biology approaches have been developed to profile the cell surface sialyation status of both in vitro and in vivo samples, including metabolic labeling, direct chemical modification, and boronic acid coupling approaches. Various investigative technologies have also been explored for their unique competence, including fluorescent imaging, flow cytometry, Raman imaging, magnetic resonance imaging (MRI), and matrix-assisted laser desorption ionization imaging mass spectrometry. In particular, the sialylation status of a specific glycoprotein on the cell surface has been investigated. This review highlights the recent advancements in chemical biology approaches for profiling cell surface sialyation status. It is expected that this review will provide researchers different choices for both biological and biomedical research and applications.

Recent approaches for directly profiling cell surface sialoform.

Sialic acids (SAs) are nine-carbon monosaccharides existing at the terminal location of glycan structures on the cell surface and secreted glycoconjugates. The expression levels and linkages of SAs on cells and tissues, collectively known as sialoform, present the hallmark of the cells and tissues of different systems and conditions. Accordingly, detecting or profiling cell surface sialoforms is very critical for understanding the function of cell surface glycans and glycoconjugates and even the molecular mechanisms of their underlying biological processes. Further, it may provide therapeutic and diagnostic applications for different diseases. In the past decades, several kinds of SA-specific binding molecules have been developed for detecting and profiling specific sialoforms of cells and tissues; the experimental materials have expanded from frozen tissue to living cells; and the analytical technologies have advanced from histochemistry to fluorescent imaging, flow cytometry and microarrays. This review summarizes the recent bioaffinity approaches for directly detecting and profiling specific SAs or sialylglycans, and their modifications of different cells and tissues.