Dynamical Effects of S_N2 Reactivity Suppression by Microsolvation: Dynamics Simulations of the F^–(H₂O) + CH₃I Reaction on a 21-Dimensional Potential Energy Surface

A comparison of atomistic dynamics between microsolvated and unsolvated reactions can expose the precise role of solvent molecules and thus provide deep insight into how solvation influences chemical reactions. Here we developed the first full-dimensional analytical potential energy surface of the F–(H2O) + CH3I reaction, which facilitates the efficient dynamics simulations on a quantitatively accurate level. The computed SN2 reactivity suppression ratio of the monosolvated F–(H2O) + CH3I reaction relative to the unsolvated F– + CH3I reaction as a function of collision energy first increases and then decreases steadily, forming an inverted-V shape, due to the combined dynamical effects of interaction time, steric hindrance, and collision-induced dehydration. Moreover, further analysis reveals that the steric effect of the F–(H2O) + CH3I reaction resulting from the single water molecule is manifested mainly in dragging the F– anion away from the central C atom, rather than shielding F– from C. Our study shows there is great potential in rigorously studying the role of the solvent in more complicated reactions

Dynamical Effects of S_N2 Reactivity Suppression by Microsolvation: Dynamics Simulations of the F^–(H₂O) + CH₃I Reaction on a 21-Dimensional Potential Energy Surface

A comparison of atomistic dynamics between microsolvated and unsolvated reactions can expose the precise role of solvent molecules and thus provide deep insight into how solvation influences chemical reactions. Here we developed the first full-dimensional analytical potential energy surface of the F–(H2O) + CH3I reaction, which facilitates the efficient dynamics simulations on a quantitatively accurate level. The computed SN2 reactivity suppression ratio of the monosolvated F–(H2O) + CH3I reaction relative to the unsolvated F– + CH3I reaction as a function of collision energy first increases and then decreases steadily, forming an inverted-V shape, due to the combined dynamical effects of interaction time, steric hindrance, and collision-induced dehydration. Moreover, further analysis reveals that the steric effect of the F–(H2O) + CH3I reaction resulting from the single water molecule is manifested mainly in dragging the F– anion away from the central C atom, rather than shielding F– from C. Our study shows there is great potential in rigorously studying the role of the solvent in more complicated reactions

Established TSC cells express markers of non-hematopoietic stem cells.

<p>(a) Representative spindle-like morphology of TSC clone 2 established from C57BL/6 E14.5 thymus repeated subculture and limiting dilution cloning.(b) Flow cytometric analysis of WT TSC with antibodies to Sca-1, CD29, CD44, CD45, CD73, CD105, CD133, CD80, MHC class I and II.</p

TSCs display thymus identity.

<p>(a) RNAs were extracted from TSC2, 1307-6.1.7 and mTEC8 cells, and transcripts were detected by RT-PCR for the expression of indicated genes. (b) Immunoblot analysis of CBX4, delta Np63, TAp63 and DNMT3a in extracts of TSC2, mTEC1 and mTEC8 cells. GAPDH was used as a loading control.</p

Established Thymic Epithelial Progenitor/Stem Cell-Like Cell Lines Differentiate into Mature Thymic Epithelial Cells and Support T Cell Development

<div><p>Common thymic epithelial progenitor/stem cells (TEPCs) differentiate into cortical and medullary thymic epithelial cells (TECs), which are required for the development and selection of thymocytes. Mature TEC lines have been widely established. However, the establishment of TEPC lines is rarely reported. Here we describe the establishment of thymic epithelial stomal cell lines, named TSCs, from fetal thymus. TSCs express some of the markers present on tissue progenitor/stem cells such as Sca-1. Gene expression profiling verifies the thymic identity of TSCs. RANK stimulation of these cells induces expression of autoimmune regulator (Aire) and Aire-dependent tissue-restricted antigens (TRAs) in TSCs <i>in vitro</i>. TSCs could be differentiated into medullary thymic epithelial cell-like cells with exogenously expressed NF-κB subunits RelB and p52. Importantly, upon transplantation under the kidney capsules of nude mice, TSCs are able to differentiate into mature TEC-like cells that can support some limited development of T cells <i>in vivo</i>. These findings suggest that the TSC lines we established bear some characteristics of TEPC cells and are able to differentiate into functional TEC-like cells <i>in vitro</i> and <i>in vivo</i>. The cloned TEPC-like cell lines may provide useful tools to study the differentiation of mature TEC cells from precursors.</p> </div

TSCs differentiate into Aire-expressing TECs <i>in vitro</i>.

<p>(a) Immunoblot analysis of Aire, delta Np63, DNMT3a, c-Myc, p52 and. RelB in extracts of TSCs stably overexpressed with p52 and RelB for 11 days. (b) Immunofluorescence analysis for UEA-1 and K8 in TSCs stably overexpressed with p52 and RelB for 11 days.</p

TSCs express cell surface markers of TEPCs.

<p>(a) Flow cytometry analysis of WT TSC line with anti-K5, anti-K8, anti-MTS24, anti-MTS10, anti-CDC205, anti-EpCAM1, 3T3 cells as a negative control for anti-CD205 and anti-EpCAM1. (b) Immunostaining of WT TSC line and 1307-6.1.7 cells with anti-K5 (green), anti-K8 (blue), anti-EpCAM1 (green), anti-Aire (red). (c) Immunostaining of WT TSC line with anti-K8 (blue) and anti-pan-cytokeratin (green).</p

Image_2_Characterization of m6A RNA Methylation Regulators Predicts Survival and Immunotherapy in Lung Adenocarcinoma.tif

N6-methyladenosine (m6A) RNA modification is a reversible mechanism that regulates eukaryotic gene expression. Growing evidence has demonstrated an association between m6A modification and tumorigenesis and response to immunotherapy. However, the overall influence of m6A regulators on the tumor microenvironment and their effect on the response to immunotherapy in lung adenocarcinoma remains to be explored. Here, we comprehensively analyzed the m6A modification patterns of 936 lung adenocarcinoma samples based on 24 m6A regulators. First, we described the features of genetic variation in these m6A regulators. Many m6A regulators were aberrantly expressed in tumors and negatively correlated with most tumor-infiltrating immune cell types. Furthermore, we identified three m6A modification patterns using a consensus clustering method. m6A cluster B was preferentially associated with a favorable prognosis and enriched in metabolism-associated pathways. In contrast, m6A cluster A was associated with the worst prognosis and was enriched in the process of DNA repair. m6A cluster C was characterized by activation of the immune system and a higher stromal cell score. Surprisingly, patients who received radiotherapy had a better prognosis than patients without radiotherapy only in the m6A cluster C group. Subsequently, we constructed an m6A score model that qualified the m6A modification level of individual samples by using principal component analysis algorithms. Patients with high m6A score were characterized by enhanced immune cell infiltration and prolonged survival time and were associated with lower tumor mutation burden and PD-1/CTLA4 expression. The combination of the m6A score and tumor mutation burden could accurately predict the prognosis of patients with lung adenocarcinoma. Furthermore, patients with high m6A score exhibited greater prognostic benefits from radiotherapy and immunotherapy. This study demonstrates that m6A modification is significantly associated with tumor microenvironment diversity and prognosis. A comprehensive evaluation of m6A modification patterns in single tumors will expand our understanding of the tumor immune landscape. In addition, our m6A score model demonstrated that the level of immune cell infiltration plays a significant role in cancer immunotherapy and provides a basis to increase the efficiency of current immune therapies and promote the clinical success of immunotherapy.</p

TSCs express Aire and tissue-restricted antigens after stimulation.

<p>(a) RT-PCR analysis for the expression of <i>aire</i>, aire-dependent <i>i-fabp</i> and aire-independent <i>crp</i> and <i>col2</i> in non-cloned WT TSC cells and cloned TSC cells (TSC2) treated with agonistic antibody to RANK (50 ng/ml) for 4 days. <i>Tubulin</i> was used as loading control. The data represented three individual experiments with similar results. (b) Quantitative PCR of mRNA expression for <i>aire</i>, <i>spt1</i> and <i>crp</i> in TSC cells treated with agonistic antibody to RANK (50 ng/ml) for 4 days. <i>Tubulin</i> was used as a reference for data normalization. Bar graphs showed means ± standard deviations of at least three independent experiments. * p < 0.05. (c and d) Immunoblot analysis of Aire in extracts of 1307-6.1.7 cell line or TSCs treated with agonistic mAb to RANK and/or agonistic mAb to LTβ receptor, TSA (0.3 µM), AZA (0.3 µM) (LTβ represents mAb to LTβ receptor; RANK represents agonistic antibody to RANK). Tubulin was used as a loading control. Data represent three independent experiments with similar results.</p

TSCs can partially support the T lymphocytes differentiation in vivo.

<p>(a) Flow cytometric analysis of thymocyte subset distribution in thymus-like tumors and corresponding spleens of nude mice 7 weeks after engraftment with re-aggregates containing thymocytes with TSCs or MEFs as defined by CD4, CD8, B220, and CD3. (b) Gross anatomy of kidneys engrafted 7 weeks earlier with MEFs (as negative control), wild type E14.5 thymus (as positive control), re-aggregates of 1×10⁴ TSCs plus MEFs or 2×10⁵ TSCs plus MEFs. (c) Flow cytometric analysis of lymphocyte subset distribution in thymus-like tumors and corresponding spleens of nude mice grafted with re-aggregates containing 1×10⁴ TSCs plus MEFs or 2×10⁵ TSCs plus MEFs as defined by CD4, CD8, B220, and CD3. (d) Frequencies of T cell populations (D3⁺ cells, CD4⁺ cells and CD8⁺ cells) in spleens of nude mice engrafted with TSC cells or MEF cells. * p < 0.05. Data represented the means ± standard deviations of three independent experiments with at least three mice per group. (e) Immunostaining of the reconstituted thymus-like tumor with anti-K8, anti-K14 and UEA-1-biotin. All data represent three individual experiments with similar results.</p