ABSTRACT
Neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being developed world over. We investigated the possibility of producing artificial antibodies from the formalin fixation and paraffin-embedding (FFPE) lung lobes of a patient who died by coronavirus disease 2019 (COVID-19). The B-cell receptors repertoire in the lung tissue where SARS-CoV-2 was detected were considered to have highly sensitive virus-neutralizing activity, and artificial antibodies were produced by combining the most frequently detected heavy and light chains. Some neutralizing effects against the SARS-CoV-2 were observed, and mixing two different artificial antibodies had a higher tendency to suppress the virus. The neutralizing effects were similar to the immunoglobulin G obtained from healthy donors who had received a COVID-19 mRNA vaccine. Therefore, the use of FFPE lung tissue, which preserves the condition of direct virus sensitization, to generate artificial antibodies may be useful against future unknown infectious diseases.
Subject(s)
COVID-19 , Humans , SARS-CoV-2 , COVID-19 Vaccines , Autopsy , Antibodies, Neutralizing , Formaldehyde , Paraffin Embedding , Receptors, Antigen, B-CellABSTRACT
Single-cell RNA-sequencing (scRNA-seq) is valuable for analyzing cellular heterogeneity. Cell composition accuracy is critical for analyzing cell-cell interaction networks from scRNA-seq data. However, droplet- and plate-based scRNA-seq techniques have cell sampling bias that could affect the cell composition of scRNA-seq datasets. Here we developed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq) for scRNA-seq based on a terminator, terminal transferase, and nanowell/bead-based scRNA-seq platform. TAS-Seq showed high tolerance to variations in the terminal transferase reaction, which complicate the handling of existing terminal transferase-based scRNA-seq methods. In murine and human lung samples, TAS-Seq yielded scRNA-seq data that were highly correlated with flow-cytometric data, showing higher gene-detection sensitivity and more robust detection of important cell-cell interactions and expression of growth factors/interleukins in cell subsets than 10X Chromium v2 and Smart-seq2. Expanding TAS-Seq application will improve understanding and atlas construction of lung biology at the single-cell level.
Subject(s)
Gene Expression Profiling , Single-Cell Analysis , Animals , DNA, Complementary/genetics , Gene Expression Profiling/methods , Humans , Mice , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , TransferasesABSTRACT
Human hepatitis B virus (HBV) infection remains a serious health problem worldwide. However, the mechanism for the maintenance of HBV in a latent state within host cells remains unclear. Here, using single-cell RNA sequencing analysis, we identified four genes linked to the maintenance of HBV in a liver cell line expressing HBV RNA at a low frequency. These genes included DOCK11 and DENND2A, which encode small GTPase regulators. In primary human hepatocytes infected with HBV, knockdown of these two genes decreased the amount of both HBV DNA and covalently closed circular DNA to below the limit of detection. Our findings reveal a role for DOCK11 and DENND2A in the maintenance of HBV.
Subject(s)
Hepatitis B/metabolism , Hepatocytes/metabolism , Host Microbial Interactions/genetics , Base Sequence , DNA, Viral/genetics , Guanine Nucleotide Exchange Factors/metabolism , Hep G2 Cells , Hepatitis B/physiopathology , Hepatitis B virus/pathogenicity , Hepatocytes/virology , Host Microbial Interactions/physiology , Humans , Latent Infection/physiopathology , Liver/pathology , Liver/virology , Primary Cell Culture , Single-Cell Analysis , Virus Replication/geneticsABSTRACT
Single cell transcriptome analysis of a cancer tissue can provide objective assessment of subtype population or the activation of each of various microenvironment component cells. In this study, we applied our newly developed technique of single cell analysis to the myometrial infiltration side (M-side) and the endometrial side (E-side) of a human endometrioid adenocarcinoma with squamous differentiation tissues. We also analyzed spherogenic cultures derived from the same tissue to identify putative regulators of stemness in vivo. Cancer cells in the E-side were highly malignant compared with those in the M-side. Many cells on the E-side were positive for spheroid-specific tumorigenesis-related markers including SOX2. In addition, there were higher numbers of epithelial-to-mesenchymal transition (EMT) cells in the E-side compared with the M-side. This study identified a site containing cells with high malignant potential such as EMT and cancer stem-like cells in cancer tissues. Finally, we demonstrate that established endometrioid adenocarcinoma subtype classifiers were variably expressed across individual cells within a tumor. Thus, such intratumoral heterogeneity may be related to prognostic implications.
