Chemoenzymatic Measurement of LacNAc in Single-Cell Multiomics Reveals It as a Cell-Surface Indicator of Glycolytic Activity of CD8+ T Cells.

Despite the rich information about the physiological state of a cell encoded in the dynamic changes of cell-surface glycans, chemical methods to capture specific glycan epitopes at the single-cell level are quite limited. Here, we report a chemoenzymatic method for the single-cell detection of N-acetyllactosamine (LacNAc) by labeling LacNAc with a specific DNA barcode. The chemoenzymatic labeling does not alter the transcriptional status of immune cells and is compatible with multiple scRNA-seq platforms. Integrated analysis of LacNAc and the transcriptome of T cells at the single-cell level reveals that the amount of cell-surface LacNAc is significantly upregulated in activated CD8+ T cells but maintained at basal levels in resting CD8+ T cells (i.e., naive and central memory T cells). Further analysis confirms that LacNAc levels are positively correlated with the glycolytic activity of CD8+ T cells during differentiation. Taken together, our study demonstrates the feasibility of the chemoenzymatic detection of cell-surface glycan in single-cell RNA sequencing-based multiomics with TCR sequence and cell-surface epitope information (i.e., scTCR and CITE-seq), and provides a new way to characterize the biological role of glycan in diverse physiological states.

Recognition of an Avr3a homologue plays a major role in mediating nonhost resistance to Phytophthora capsici in Nicotiana species.

Nonhost resistance is a commonly occurring phenomenon wherein all accessions or cultivars of a plant species are resistant to all strains of a pathogen species and is likely the manifestation of multiple molecular mechanisms. Phytophthora capsici is a soil-borne oomycete that causes Phytophthora blight disease in many solanaceous and cucurbitaceous plants worldwide. Interest in P. capsici has increased considerably with the sequencing of its genome and its increasing occurrence in multiple crops. However, molecular interactions between P. capsici and both its hosts and its nonhosts are poorly defined. We show here that tobacco (Nicotiana tabacum) acts like a nonhost for P. capsici and responds to P. capsici infection with a hypersensitive response (HR). Furthermore, we have found that a P. capsici Avr3a-like gene (PcAvr3a1) encoding a putative RXLR effector protein produces a HR upon transient expression in tobacco and several other Nicotiana species. This HR response correlated with resistance in 19 of 23 Nicotiana species and accessions tested, and knock-down of PcAvr3a1 expression by host-induced gene silencing allowed infection of resistant tobacco. Our results suggest that many Nicotiana species have the capacity to recognize PcAvr3a1 via the products of endogenous disease resistance (R) genes and that this R gene-mediated response is a major component of nonhost resistance to P. capsici.

Biocompatible click chemistry enabled compartment-specific pH measurement inside E. coli.

Bioorthogonal reactions, especially the Cu(I)-catalysed azide-alkyne cycloaddition, have revolutionized our ability to label and manipulate biomolecules under living conditions. The cytotoxicity of Cu(I) ions, however, has hindered the application of this reaction in the internal space of living cells. By systematically surveying a panel of Cu(I)-stabilizing ligands in promoting protein labelling within the cytoplasm of Escherichia coli, we identify a highly efficient and biocompatible catalyst for intracellular modification of proteins by azide-alkyne cycloaddition. This reaction permits us to conjugate an environment-sensitive fluorophore site specifically onto HdeA, an acid-stress chaperone that adopts pH-dependent conformational changes, in both the periplasm and cytoplasm of E. coli. The resulting protein-fluorophore hybrid pH indicators enable compartment-specific pH measurement to determine the pH gradient across the E. coli cytoplasmic membrane. This construct also allows the measurement of E. coli transmembrane potential, and the determination of the proton motive force across its inner membrane under normal and acid-stress conditions.

Click Triazoles for Bioconjugation.

Click Chemistry is a set of rapid, selective and robust reactions that give near-quantitative yield of the desired product in aqueous solutions. The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) that forms 1,4-disubstituted triazoles is a prototypical example of click chemistry that features exquisite selectivity and bioorthogonality-that is, non-interacting with biological components while proceeding under physiological conditions. Over the past ten years, CuAAC has found extensive applications in the field of chemical biology. In this chapter, we describe the discovery of Cu(I) catalysts for this transformation and the recent development of the strain-promoted azide-alkyne cycloaddition that eliminate the use of copper. We also highlight several recent applications toward conjugating biomolecules, including proteins, nucleic acids, lipids and glycans, with biophysical probes for both in vitro and in vivo studies.

QKI-mediated alternative splicing of the histone variant MacroH2A1 regulates cancer cell proliferation.

The histone variant macroH2A1 contains a carboxyl-terminal ∼30-kDa domain called a macro domain. MacroH2A1 is produced as one of two alternatively spliced forms, macroH2A1.1 and macroH2A1.2. While the macro domain of macroH2A1.1 can interact with NAD(+)-derived small molecules, such as poly(ADP-ribose), macroH2A1.2's macro domain cannot. Here, we show that changes in the alternative splicing of macroH2A1 pre-mRNA, which lead to a decrease in macroH2A1.1 expression, occur in a variety of cancers, including testicular, lung, bladder, cervical, breast, colon, ovarian, and endometrial. Furthermore, reintroduction of macroH2A1.1 suppresses the proliferation of lung and cervical cancer cells in a manner that requires the ability of macroH2A1.1 to bind NAD(+)-derived metabolites. MacroH2A1.1-mediated suppression of proliferation occurs, at least in part, through the reduction of poly(ADP-ribose) polymerase 1 (PARP-1) protein levels. By analyzing publically available expression and splicing microarray data, we identified splicing factors that correlate with alterations in macroH2A1 splicing. Using RNA interference, we demonstrate that one of these factors, QKI, regulates the alternative splicing of macroH2A1 pre-mRNA, resulting in increased levels of macroH2A1.1. Finally, we demonstrate that QKI expression is significantly reduced in many of the same cancer types that demonstrate a reduction in macroH2A1.1 splicing.