No abstract

Autophagy modulators are considered putative therapeutic targets because of the role of autophagy in cancer progression. Kazinol C, a 1,3-diphenylpropane from the plant Broussonetia kazinoki , has been shown to induce apoptosis in colon cancer cells through the activation of AMPK at high concentrations. In the present study, we found that Kazinol C induced autophagy through endoplasmic reticulum stress-mediated unfolded protein response signaling in several normal and cancer cell lines at low concentrations of Kazinol C that did not induce apoptosis. Kazinol C activated the transducers of unfolded protein response signaling, leading to target gene expression, LC3-II conversion, and TFEB nuclear translocation. Chemical inhibition of endoplasmic reticulum stress reduced LC3-II conversion. In addition, blockade of autophagy by knockout of Atg5 or treatment with 3-MA enhanced Kazinol C-induced apoptosis. In summary, we have uncovered Kazinol C as a novel autophagy inducer and confirmed the role of autophagy as a cellular stress protector.

γδ T cells have the potential for adoptive immunotherapy since they respond to bacteria, viruses, and tumors. However, these cells represent a small fraction of the peripheral T-cell pool and require activation and proliferation for clinical benefits. In cord blood, there are some γδ T cells, which exhibit a naïve phenotype, and mostly include Vδ1+ T cells. In this study, we investigated the effect of CD3 signaling on cord blood γδ T-cell proliferation using K562-based artificial antigen presenting cells expressing costimulatory molecules. There were significantly more Vδ1+ T cells in the group stimulated with anti-CD3 antibody than in the group without. In cultured Vδ1+ T cells, DNAM-1 and NKG2D were highly expressed, but NKp30 and NKp44 showed low expression. Among various target cells, Vδ1+ T cells showed the highest cytotoxicity against U937 cells, but Daudi and Raji cells were not susceptible to Vδ1+ T cells. The major cytokines secreted by Vδ1+ T cells responding to U937 cells were Granzyme B, IFN-γ, and sFasL. Cytotoxicity by Vδ1+ T cells correlated with the expression level of PVR and Nectin of DNAM-1 ligands on the surface of target cells. Compared to Vδ2+ T cells in peripheral blood, cord blood Vδ1+ T cells showed varying cytotoxicity patterns depending on the target cells. Here, we determined the ideal conditions for culturing cord blood Vδ1+ T cells by observing that Vδ1+ T cells were more sensitive to CD3 signals than other subtypes of γδ T cells in cord blood. Cultured cord blood Vδ1+ T cells recognized target cells through activating receptors and secreted numerous cytotoxic cytokines. These results are useful for the development of tumor immunotherapy based on γδ T cells.

Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a revolutionary genomic mapping strategy developed by the group of Dr. Steven Henikoff. In CUT&RUN, cells or nuclei are immobilized to a solid support, with pAG-MNase cleaved DNA fragments isolated from solution. The workflow is compatible with next-generation sequencing (NGS) to provide high quality genome-wide profiles of histone post-translational modifications (PTMs) and chromatin-associated proteins (e.g. TFs and chromatin remodelers) Historically, ChIP-seq is the leading approach for genome-wide mapping of histone PTMs and chromatin-associated proteins. ChIP-seq requires large numbers of cells (typically 105 - 106 cells) and deep sequencing of both input chromatin and immunoprecipitated material (typically > 30 million read each) to resolve signal from background. With this innovation, background is dramatically reduces, allowing high resolution genomic mapping for histone PTMs and chromatin-associated proteins using a small number of cells and only 3-8 million sequencing reads per sample. Although it is recommended to start with 500,000 cells, comparable data can be generated using as few as 5,000 cells. Overview of the CUTANA CUT&RUN protocol 1. Immobilize & permeabilize cells (or nuclei) 2. Add antibody to histone PTM or chromatin-interacting protein 3. Add & activate pAG-MNase to cleave target-DNA complex 4. Target-DNA complex diffuses out, collect supernatant 5. Extract DNA & prepare sequencing library 6. Next-generation sequencing and data analysis from CUTANA CUT&RUN kit manual

No abstract