Droplet-Based Glycan and RNA Sequencing for Profiling the Distinct Cellular Glyco-States in Single Cells.

Plate-based single-cell glycan and RNA sequencing (scGR-seq) is previously developed to realize the integrated analysis of glycome and transcriptome in single cells. However, the sample size is limited to only a few hundred cells. Here, a droplet-based scGR-seq is developed to address this issue by adopting a 10x Chromium platform to simultaneously profile ten thousand cells' glycome and transcriptome in single cells. To establish droplet-based scGR-seq, a comparative analysis of two distinct cell lines is performed: pancreatic ductal adenocarcinoma cells and normal pancreatic duct cells. Droplet-based scGR-seq revealed distinct glycan profiles between the two cell lines that showed a strong correlation with the results obtained by flow cytometry. Next, droplet-based scGR-seq is applied to a more complex sample: peripheral blood mononuclear cells (PBMC) containing various immune cells. The method can systematically map the glycan signature for each immune cell in PBMC as well as glycan alterations by cell lineage. Prediction of the association between the glycan expression and the gene expression using regression analysis ultimately leads to the identification of a glycan epitope that impacts cellular functions. In conclusion, the droplet-based scGR-seq realizes the high-throughput profiling of the distinct cellular glyco-states in single cells.

Ionization and electron excitation of C60 in a carbon nanotube: A variable temperature/voltage transmission electron microscopic study.

There is increasing attention to chemical applications of transmission electron microscopy, which is often plagued by radiation damage. The damage in organic matter predominantly occurs via radiolysis. Although radiolysis is highly important, previous studies on radiolysis have largely been descriptive and qualitative, lacking in such fundamental information as the product structure, the influence of the energy of the electrons, and the reaction kinetics. We need a chemically well-defined system to obtain such data and have chosen as a model a variable-temperature and variable-voltage (VT/VV) study of the [2 + 2] dimerization of a van der Waals dimer [60]fullerene (C60) to C120 in a carbon nanotube (CNT), as studied for several hundred individual reaction events at atomic resolution. We report here the identification of five reaction pathways that serve as mechanistic models of radiolysis damage. Two of them occur via a radical cation of the specimen generated by specimen ionization, and three involve singlet or triplet excited states of the specimen, as initiated by electron excitation of the CNT, followed by energy transfer to the specimen. The [2 + 2] product was identified by measuring the distance between the two C60 moieties, and the mechanisms were distinguished by the pre-exponential factor and the Arrhenius activation energy­the standard protocol of chemical kinetic studies. The results illustrate the importance of VT/VV kinetic analysis in the studies of radiation damage and show that chemical ionization and electron excitation are inseparable, but different, mechanisms of radiation damage, which has so far been classified loosely under the single term "ionization."

Direct Microscopic Analysis of Individual C60 Dimerization Events: Kinetics and Mechanisms.

Modern transition state theory states that the statistical behavior of a chemical reaction is the sum of individual chemical events that occur randomly. Statistical analysis of each event for individual molecules in a three-dimensional space however is practically impossible. We report here that kinetics and mechanisms of chemical reactions can be investigated by using a one-dimensional system where reaction events can be observed in situ and counted one by one using variable-temperature (VT) atomic-resolution transmission electron microscopy (TEM). We thereby provide direct proof that the ensemble behavior of random events conforms to the Rice-Ramsperger-Kassel-Marcus theory, as illustrated for [2 + 2] cycloaddition of C60 molecules in carbon nanotubes (CNTs). This method gives kinetic and structural information for different types of reactions occurring simultaneously in the microscopic view field, suggesting that the VT-TEM opens a new dimension of chemical kinetics research on molecules and their assemblies in their excited and ionized states. The study carried out at 393-493 K showed that pristine CNT primarily acts as a singlet sensitizer of the cycloaddition reaction that takes place with an activation energy of 33.5 ± 6.8 kJ/mol. On the other hand, CNT suffers electron damage of the conjugated system at 103-203 K and promotes a reactive radical cation path that takes place with an activation energy of only 1.9 ± 0.7 kJ/mol. The pre-exponential factor of the Arrhenius plot gave us further mechanistic insights.