Aminolysis of a Model Nerve Agent: A Computational Reaction Mechanism Study of <i>O</i>,<i>S</i>‑Dimethyl Methylphosphonothiolate

The mechanism for the aminolysis of a model nerve agent, <i>O</i>,<i>S</i>-dimethyl methylphosphonothiolate, is investigated both at density functional level using M062X method with 6-311++G­(d,p) basis set and at ab initio level using the second-order Møller–Plesset perturbation theory (MP2) with the 6-311+G­(d,p) basis set. The catalytic role of an additional NH₃ and H₂O molecule is also examined. The solvent effects of acetonitrile, ethanol, and water are taken into account employing the conductor-like screening model (COSMO) at the single-point M062X/6-311++G­(d,p) level of theory. Two possible dissociation pathways, methanethiol and methyl alcohol dissociations, along with two different neutral mechanisms, a concerted one and a stepwise route through two neutral intermediates, for each pathway are investigated. Hyperconjugation stabilization that has an effect on the stability of generated transition states are investigated by natural bond order (NBO) approach. Additionally, quantum theory of atoms in molecules analysis is performed to evaluate the bond critical (BCP) properties and to quantify strength of different types of interactions. The calculated results predict that the reaction of <i>O</i>,<i>S</i>-dimethyl methylphosphonothiolate with NH₃ gives rise to parallel P–S and P–O bond cleavages, and in each cleavage the neutral stepwise route is always favorable than the concerted one. The mechanism of NH₃ and H₂O as catalyst is nearly similar, and they facilitate the shuttle of proton to accelerate the reaction. The steps involving the H₂O-mediated proton transfer are the most suitable ones. The first steps for the stepwise process, the formation of neutral intermediate, are the rate-determining step. It is observed that in the presence of catalyst the reaction in the stepwise path possesses almost half the activation energy of the uncatalyzed one. A bond-order analysis using Wiberg bond indexes obtained by NBO calculation predicts that usually all individual steps of the reactions occur in a concerted fashion showing equal progress along different reaction coordinates

Comprehensive Study of Methylation on the Silicon (100)‑2 × 1 Surface: A Density Functional Approach

A detailed mechanistic investigation of Si–Me formation over the silicon (100)-2 × 1 surface using the Si₉H₁₂ cluster model has been performed using various reagents, based on two basic mechanisms: dissociation and substitution. The reagents CH₄, CH₃Cl for dissociation and CH₃Li, CH₃MgBr for substitution mechanism are used to explore the methylation process on the silicon surface at the M062X/6-311+G­(2d, p) level of theory. The associated potential energy surfaces explored here are aimed to unveil the most favored pathway of methylation with appropriate reagents. Dissociation of methane forms a monomethylated product (D1) through an energetically unfavorable pathway. All the adsorption modes of CH₃Cl over the silicon surface are also detected and analyzed. Methyl chloride dissociates to form another monomethylated product D2 and its derivative D3 in the entrance channel, while, in the next step, bridged compounds I1 (Cl-bridged) and I2 (H-bridged) are produced from them, respectively. The C–Cl dissociation leads to the formation of D2 having a lower activation barrier. With a comparably high activation barrier in the C–H dissociation, producing D3, very interestingly carbene intermediate has been detected in the reaction pathway. Detection of energetically unfavored conversions from D2 to I1 and D3 to I2 ensured that the methylation process will not be hampered through these interconversions. For substitution, HCl- and Cl₂-passivated Si surfaces are taken, where chlorine is to be substituted by the methyl group of both of the methylating agents. With both substituents, HCl-passivated Si₉H₁₂ gives D1. The substitution process on Cl₂-passivated Si₉H₁₂ leads to the formation of D2 in the first step and dimethylated product (S1) in the final step. In all the above substitution processes, methyl lithium proved to be the better substituent for the formations of D1, D2, and S1 on HCl- or Cl₂-passivated surfaces. The present work not only demonstrated methyl lithium as one of the best methylating agents but also revealed the interrelation among the dissociative adsorption modes of CH₃Cl, reported earlier, in a single potential energy surface with a remarkable detection of carbene intermediate formed in the pathway of C–H dissociation

Uncropped western raw images.

Figs 1F, 1G, 2A, 2I, 2N, 4D, 4G, 4I, 5A, 5B, 5D, 5E, 5H, 5J, 5L, 6B, 6H, 6K, S4D, S4F, S5A, S5E, S5M, S5K, S5O, and S6H. (PDF)</p

NCoR1 controls the mTOR-TFEB axis to regulate autophagy and lysosome biogenesis in myeloid cells.

(A, B) Representative western blot image with corresponding densitometric analysis depicting the TFEB protein kinetics (2 h, 12 h, and 24 h) in the H37Rv infected cDC1. All protein bands were normalised with β-actin housekeeping control (n = 3). (C, D) Microscopy images showing the relative levels of NCoR1 and TFEB levels in CD11c+ and F4/80+ H37Rv-GFP infected lung tissue sections compared to uninfected C57BL/6 mice. (E, F) Western blot image and bar plot demonstrating the TFEB level in control and NCoR1 KD cDC1 at different time points upon H37Rv infection (n = 3). (G) Confocal microscopy showing NCoR1 and TFEB expression in H37Rv infected BMDMs generated from NCoR1MyeKO and NCoR1fl/fl mice (n = 4 mice). (H) Bar plot showing the quantification for NCoR1 and TFEB protein levels from confocal microscopy of H37Rv infected BMDMs generated from NCoR1MyeKO and NCoR1fl/fl mice (n = 4 mice). (I, J) Confocal microscopy images and bar plots showing the entrapment of H37Rv with LAMP1 protein in control and NCoR1 KD human monocytic THP1 differentiated macrophages at different time points (n = 3). (K) Western blot image showing the protein levels of NCoR1, TFEB, and LC3-II:LC3-I in starved and fed condition in control and NCoR1 KD human monocytic THP-1 differentiated macrophages (n = 3). (L) Bar plot showing densitometric quantification of NCoR1, TFEB, and LC3-II:LC3-I western bands in starved and fed condition in control and NCoR1 KD human monocytic THP-1 differentiated macrophages. All bands were normalised with β-actin as housekeeping control (n = 3). (M) Western blot image showing NCoR1 and LC3-II:LC3-I protein levels in control and NCoR1 KD human monocytic THP-1 differentiated macrophages treated with heat killed H37Rv at different time points (n = 3). (N) Bar plot depicting densitometric quantification of NCoR1 and LC3-II:LC3-I in control and NCoR1 KD human monocytic THP-1 differentiated macrophages treated with heat killed H37Rv at different time points. All bands were normalised with β-actin as housekeeping control (n = 3). (O) Western blot representative image depicting the p-mTOR, mTOR, and p-TFEB, TFEB levels in H37Rv infected control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 2 h and 24 h post infection, with and without Torin1 treatment (n = 3). (P) Bar plot depicting the densitometric quantification of normalised p-mTOR and p-TFEB protein bands. The p-mTOR and p-TFEB levels were normalised first with their respective total protein levels and then with housekeeping control β-actin, with and without Torin1 (n = 3). (Q) FACS analysis demonstrating the MFI shifts for H37Rv infection in control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 24 h of infection with and without treatment of rapamycin (n = 3). (R) Bar plot demonstrating the MFI shift quantification of H37Rv infection in control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 24 h of infection with and without treatment of rapamycin (n = 3). *p p p t test. Where n represents independent biological replicates. The data underlying this figure are available in S1 Data. Western blot raw images can be found in S1 Raw Image. (TIF)</p

NCoR1 controls the autophagy induction in both human and murine myeloid cells upon <i>Mycobacterium</i> infection.

(A) Flow cytometry contour plots showing the intracellular H37Rv bacterial load in control and NCoR1 KD human monocytic THP-1 differentiated macrophages, with and without bafilomycin treatment (n = 3). (B, C) Flow cytometry histograms showing the MFI shifts for the H37Rv infection in control and NCoR1 KD human monocytic THP-1 differentiated macrophages with and without treatment of bafilomycin, bar plots depicting the quantification of the same (n = 3). (D) Western blot image showing the NCoR1 and LC3-II:LC3-I protein levels in control and NCoR1 KD cDC1 at different time points upon H37Rv infection (n = 3). (E) Bar plot showing densitometric quantification for the NCoR1 and LC3-II:LC3-I levels in control and NCoR1 KD cDC1 at different time points upon H37Rv infection. All protein bands were normalised with β-actin housekeeping control (n = 3). (F) Western blot image and corresponding densitometric analysis demonstrating the LC3-II:LC3-I protein levels in control and NCoR1 KD THP-1 differentiated mo-mΦ upon H37Rv infection vs. uninfected (n = 3). (G, H). Confocal microscopy and corresponding bar plot demonstrating the colocalization of H37Rv with LC3 protein in the BMDMs from NCoR1fl/fl and NCoR1MyeKO mice at 2 h and 24 h post infection (n = 4 mice). *p p p t test, where n represents independent biological replicates. The data underlying this figure are available in S1 Data. Western blot raw images can be found in S1 Raw Image. (TIF)</p

Numerical values for all datasets.

Figs 1A–1C, 1E–1G, 2B–2C, 2E, 2G–2H, 2J–2M, 2O–2R, 3A–3K, 4A–4C, 4E–4F, 4H, 4J, 4L, 5A–5G, 5I, 5K, 5M, 5N, 6A, 6C–6J, 6L, 6M, S1A–S1B, S2A–S2B, S2E, S2G–S2H, S2J, S2L–S2N, S2O, S4C, S4E–S4F, S4H, S5B, S5F, S5H, S5J, S5, S5L, S5N, S5P, S5R, S6A–S6D, S6G, S6I, and S6J. (XLSX)</p

Myeloid specific NCoR1 deletion exacerbates <i>Mycobacterium</i> infection in mice.

(A) Line graph showing the percent reduction in body weight upon H37Rv infection in NCoR1fl/fl and NCoR1MyeKO mice at regular intervals till day 21 post infection (n = 5 mice). (B, C) Bar plots showing the bacterial load in the lung tissues of H37Rv-infected NCoR1fl/fl and NCoR1MyeKO mice at day 7 and day 21 post infection by CFU assay in lung and spleen. Data is presented as the median log10CFU (n = 4–5). (D) Bar plots showing the percent positive myeloid cell subtypes gated on CD45 positive cells isolated from lung tissues of NCoR1fl/fl and NCoR1MyeKO mice on day 21 post infection. Strategy used to gate H37Rv-infected macrophages in FACS is shown in S3C Fig (n = 5). (E) Bar plots showing the percent positive myeloid cell subtypes gated on CD45 positive cells isolated from spleen tissues of NCoR1fl/fl and NCoR1MyeKO mice on day 21 post infection. Strategy used to gate H37Rv-infected macrophages in FACS is shown in S3D Fig (n = 5). (F) Bar plots showing the percentage of GFP-tagged H37Rv infection in neutrophils, alveolar macrophages, dendritic cells, eosinophils, infiltrating macrophages, and inflammatory monocytes, and (G) corresponding MFI shifts in the cells isolated from lung tissues of NCoR1fl/fl and NCoR1MyeKO mice on day 21 post infection (n = 5). (H) Bar plot showing the percentage of GFP-tagged H37Rv infection in dendritic cells, macrophages, monocytes, and neutrophils, and (I) corresponding MFI shifts in the cells isolated from spleen tissues of NCoR1fl/fl and NCoR1MyeKO mice on day 21 post infection (n = 4). (J) Bar plot showing the percent positive B cell and T cell subtypes gated on CD45 positive cells isolated from splenic tissues of NCoR1fl/fl and NCoR1MyeKO mice on day 21 post infection. Strategy used to gate H37Rv-infected macrophages in FACS is shown in S3E Fig (n = 5). (K) Bar plot showing the level of different inflammatory cytokines in the lung tissue lysate of NCoR1fl/fl and NCoR1MyeKO mice on day 21, normalised to protein content (n = 5). (L) Representative HE staining image showing infiltration of cells in the lung tissue of NCoR1fl/fl and NCoR1MyeKO mice on day 21. *p p p t test. Where n represents the total number of used mice. The data underlying this figure are available in S1 Data. HE, haematoxylin and eosin; MFI, mean fluorescence intensity.</p

NCoR1 regulates mTOR-TFEB axis to control autophagy induction and lysosomal biogenesis in myeloid cells.

(A) Representative western blot image along with bar plots for densitometric analysis depicting the TFEB protein kinetics (2 h, 12 h, and 24 h) in the H37Rv-infected human monocytic THP-1 differentiated macrophages. For normalisation, β-actin was used as housekeeping control (n = 3). (B) Western blot image depicting the levels of TFEB protein in H37Rv-infected control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 2 h, 12 h, and 24 h post infection. Corresponding bar plots showing the densitometric analysis from 3 independent biological replicates. For normalisation, β-actin was used as housekeeping control (n = 3). (C) Violin plot depicting the normalised transcript expression of TFEB in RNA-seq data of control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 2 h and 24 h post infection (n = 3). (D) Representative western blot image with densitometric analysis showing the TFEB and LAMP1 levels in the BMDMs from NCoR1fl/fl and NCoR1MyeKO mice at 2 h and 24 h post H37Rv infection. For normalisation, β-actin was used as housekeeping control (n = 4 mice). (E) Western blot image along with densitometric analysis showing the levels of LC3, LAMP1, and TFEB-flag in H37Rv-infected NCoR1 KD human monocytic THP-1 differentiated macrophages with or without overexpression of exogenous flag-tagged TFEB at 24 h post infection. For normalisation, β-actin was used as housekeeping control (n = 3). (F) Scatter plot demonstrating the H37Rv bacterial load by CFU assay in H37Rv-infected NCoR1 KD human monocytic THP-1 differentiated macrophages with or without exogenous overexpression of flag-tagged TFEB at 24 h post infection (n = 4). (G) Heat map showing the DEGs related to mTOR pathway in RNA-seq data of control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 2 h and 24 h post infection (n = 3). (H) Representative western blot image depicting the kinetics (2 h, 12 h, 24 h) of phospho-mTOR (p-mTOR), mTOR, phospho-TFEB (p-TFEB), TFEB, and LC3-II:LC3-I protein levels in H37Rv-infected control and NCoR1 depleted human monocytic THP-1 differentiated macrophages (n = 3). (I) Bar plot showing the densitometric quantification of p-mTOR, mTOR, p-TFEB, TFEB, and LC3 protein bands from 3 independent biological replicates in H37Rv-infected human control and NCoR1 KD monocytic THP-1 differentiated macrophages at 2 h, 12 h, and 24 h post infection. p-mTOR and p-TFEB were normalised first with total protein levels and then with housekeeping control β-actin. LC3-II density versus LC3-I was quantified followed by normalisation with β-actin (n = 3). (J) Representative western blot image depicting the protein levels of p-mTOR and total mTOR in H37Rv-infected BMDMs generated from NCoR1fl/fl and NCoR1MyeKO mice at 2 h and 24 h post infection. For normalisation, β-actin was used as housekeeping control (n = 4 mice). (K) Bar plot showing the densitometric quantification of p-mTOR levels from 3 independent biological replicates in H37Rv-infected BMDMs generated from NCoR1fl/fl and NCoR1MyeKO mice at 2 h and 24 h post infection. p-mTOR was normalised first with total m-TOR followed by normalisation with β-actin (n = 4 mice). (L) Western blot representative image depicting the p-mTOR, mTOR, p-TFEB, TFEB, and LC3-II:LC3-I levels in H37Rv-infected control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 2 h and 24 h post infection, with and without rapamycin treatment (n = 3). (M) Bar plot depicting the densitometric quantification of normalised p-mTOR, p-TFEB, and LC3 protein bands from 3 independent biological replicates. The p-mTOR and p-TFEB levels were normalised first with their respective total protein levels and then with housekeeping control β-actin. LC3-II versus LC3-I levels were quantified followed by normalisation with β-actin (n = 3). (N) Scatter plot showing the bacterial load in H37Rv-infected control and NCoR1 KD human monocytic THP-1 differentiated macrophages by CFU assay at 24 h post infection, with and without treatment of rapamycin (n = 4). *p p p t test. Where n represents independent biological replicates. The data underlying this figure are available in S1 Table and S1 Data. Western blot raw images can be found in S1 Raw Image. BMDM, bone marrow-derived macrophage; DEG, differentially expressed gene; KD, knockdown; TFEB, transcription factor EB.</p

Graphical abstract.

Mycobacterium tuberculosis (Mtb) defends host-mediated killing by repressing the autophagolysosome machinery. For the first time, we report NCoR1 co-repressor as a crucial host factor, controlling Mtb growth in myeloid cells by regulating both autophagosome maturation and lysosome biogenesis. We found that the dynamic expression of NCoR1 is compromised in human peripheral blood mononuclear cells (PBMCs) during active Mtb infection, which is rescued upon prolonged anti-mycobacterial therapy. In addition, a loss of function in myeloid-specific NCoR1 considerably exacerbates the growth of M. tuberculosis in vitro in THP1 differentiated macrophages, ex vivo in bone marrow-derived macrophages (BMDMs), and in vivo in NCoR1MyeKO mice. We showed that NCoR1 depletion controls the AMPK-mTOR-TFEB signalling axis by fine-tuning cellular adenosine triphosphate (ATP) homeostasis, which in turn changes the expression of proteins involved in autophagy and lysosomal biogenesis. Moreover, we also showed that the treatment of NCoR1 depleted cells by Rapamycin, Antimycin-A, or Metformin rescued the TFEB activity and LC3 levels, resulting in enhanced Mtb clearance. Similarly, expressing NCoR1 exogenously rescued the AMPK-mTOR-TFEB signalling axis and Mtb killing. Overall, our data revealed a central role of NCoR1 in Mtb pathogenesis in myeloid cells.</div

NCoR1 regulates autophagy induction during <i>Mtb</i> infection.

(A) Heat map showing the top differential expressed genes related to autophagy function in the RNA-seq data of control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 2 h and 24 h post infection (n = 3). (B) Violin plot depicting the normalised transcript expression of ATGs in control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 2 h and 24 h post infection (n = 3). (C) String network analysis showing the association of NCoR1 with top DEGs found in NCoR1 KD human monocytic THP-1 differentiated macrophages vs. control cells at 2 h and 24 h post infection (n = 3). (D, E) Representative western blot image depicting the LC3-II:LC3-I level in H37Rv-infected control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 2 h and 24 h post infection, before and after treatment with bafilomycin. Corresponding densitometric analysis (bar plots) depicting the quantitation and statistics from the western blot images of 3 independent biological replicates. For normalisation, β-actin was used as housekeeping control. LC3-II density versus LC3-I was quantified followed by normalisation with β-actin (n = 3). (F) Scatter plot showing the H37Rv bacterial load in control and NCoR1 KD human monocytic THP-1 differentiated macrophages by CFU assay at 24 h post infection, before and after treatment with bafilomycin (n = 4). (G, H) Western blot representative image depicting the protein levels of Beclin1 and ATG12-5 in H37Rv-infected control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 2 h and 24 h post infection. Corresponding densitometric analysis (bar plots) showing the quantitation and statistics from the western blot images of 3 independent biological replicates. For normalisation, β-actin was used as housekeeping control (n = 3). (I) Western blot representative picture showing the levels of NCoR1, Atg12-Atg5, Beclin1, and LC3-II:LC3-I in H37Rv-infected BMDMs generated from NCoR1fl/fl and NCoR1MyeKO mice at 2 h and 24 h post infection. For normalisation, β-actin was used as housekeeping control. LC3-II density versus LC3-I was quantified followed by normalisation with β-actin (n = 4 mice). (J) Bar plot showing the densitometric quantification from western blot images for NCoR1, ATG12-ATG5, BECLIN1, and LC3 in H37Rv-infected BMDMs generated from NCoR1MyeKO and NCoR1fl/fl mice at 2 h and 24 h post infection. For normalisation, β-actin was used as housekeeping control (n = 4 mice). (K) Confocal microscopy showing the colocalization of H37Rv with LC3 protein in control and NCoR1 KD human monocytic THP-1 differentiated macrophages at 2 h and 24 h post infection (n = 3). (L) Bar plot depicting the quantification of confocal images from 3 independent biological replicates for the colocalization of H37Rv with LC3 in control and NCoR1 KD human monocytic THP-1 differentiated macrophages. Ten cells from each biological replicate were analysed for calculating the colocalization percentage (n = 3). *p p p t test. Where n represents independent biological replicates. The data underlying this figure are available in S1 Table and S1 Data. Western blot raw images can be found in S1 Raw Image. BMDM, bone marrow-derived macrophage; DEG, differentially expressed gene; KD, knockdown.</p