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Monitoring the build-up or decay of hyperpolarization in nuclear magnetic resonance requires radio-frequency (RF) pulses to generate observable nuclear magnetization. However, the pulses also lead to a depletion of the polarization and, thus, alter the spin dynamics. To simulate the effects of RF pulses on the polarization build-up and decay, we propose a first-order rate-equation model describing the dynamics of the hyperpolarization process through a single source and a relaxation term. The model offers a direct interpretation of the measured steady-state polarization and build-up time constant. Furthermore, the rate-equation model is used to study three different methods to correct the errors introduced by RF pulses: (i) a 1/cos⁡n-1θ correction (θ denoting the RF pulse flip angle), which is only applicable to decays; (ii) an analytical model introduced previously in the literature; and (iii) an iterative correction approach proposed here. The three correction methods are compared using simulated data for a range of RF flip angles and RF repetition times. The correction methods are also tested on experimental data obtained with dynamic nuclear polarization (DNP) using 4-oxo-TEMPO in 1H glassy matrices. It is demonstrated that the analytical and iterative corrections allow us to obtain accurate build-up times and steady-state polarizations (enhancements) for RF flip angles of up to 25∘ during the polarization build-up process within ±10 % error when compared to data acquired with small RF flip angles (∘). For polarization decay experiments, corrections are shown to be accurate for RF flip angles of up to 12∘. In conclusion, the proposed iterative correction allows us to compensate for the impact of RF pulses offering an accurate estimation of polarization levels, build-up and decay time constants in hyperpolarization experiments.

Fast magic-angle spinning (MAS) NMR experiments open the way for proton-detected NMR studies and have been explored in the past years for a broad range of materials, comprising biomolecules and pharmaceuticals. Proton-spin diffusion (SD) is a versatile polarization-transfer mechanism and plays an important role in resonance assignment and structure determination. Recently, the occurrence of negative cross peaks in 2D 1H-1H SD-based spectra has been reported associated with higher-order SD effects, in which the chemical shifts of the involved quadruple of nuclei need to compensate each other. We herein report negative cross peaks in SD-based spectra observed for a variety of small organic molecules involving methyl groups. We combine experimental observations with numerical and analytical simulations to demonstrate that the methyl groups can give rise to coherent (SD) as well as incoherent (Nuclear Overhauser Enhancement, NOE) effects, both in principle manifesting themselves as negative cross peaks in the 2D spectra. The simulations however reveal that higher order coherent contributions dominate the experimentally observed negative peaks. Methyl groups are prone to the observation of such higher order coherent effects. Due to their low-frequency shifted 1H resonances, the chemical-shift separation relative to for instance aromatic protons in spatial proximity is substantial (> 4.7 ppm in the studied examples) preventing any sizeable second-order spin-diffusion processes, which would superimpose the negative contribution to the peaks.

Despite many decades of research, homonuclear decoupling in solid-state NMR under magic-angle spinning (MAS) has yet to reach a point where the achievable proton line widths become comparable to the resolution obtained in solution-state NMR. This makes the precise determination of isotropic chemical shifts difficult and thus presents a limiting factor in the application of proton solid-state NMR to biomolecules and small molecules. In this publication we analyze the sources of the residual line width in refocused homonuclear-decoupled spectra in detail by comparing numerical simulations and experimental data. Using a hybrid analytical/numerical approach based on Floquet theory, we find that third-order effective Hamiltonian terms are required to realistically characterize the line shape and line width under frequency-switched Lee-Goldburg (FSLG) decoupling under MAS. Increasing the radio-frequency field amplitude enhances the influence of experimental rf imperfections such as pulse transients and the MAS-modulated radial rf-field inhomogeneity. While second- and third-order terms are, as expected, reduced in size at higher rf-field amplitudes, the line width becomes dominated by first-order terms which severely limits the achievable line width. We expect, therefore, that significant improvements in the line width of FSLG-decoupled spectra can only be achieved by reducing the influence of MAS-modulated rf-field inhomogeneity and pulse transients., Physical Chemistry Chemical Physics, 25 (17), ISSN:1463-9084, ISSN:1463-9076

Adenomatous polyposis coli (APC) truncations occur in many colorectal cancers and are often associated with immune infiltration. The aim of this study was to determine whether a combination of Wnt inhibition with anti-inflammatory (sulindac) and/or proapototic (ABT263) drugs can reduce colon adenomas. Apcmin/+ and doublecortin-like kinase 1 (Dclk1)Cre/+;Apcfl/fl mice were exposed to dextran sulphate sodium (DSS) in their drinking water to promote the formation of colon adenomas. Mice were then treated with either a Wnt-signaling antagonist pyrvinium pamoate (PP), an anti-inflammatory agent sulindac or proapoptotic compound ABT263 or a combination of PP+ABT263, or PP+sulindac. Colon adenoma frequency, size, and T-cell abundance were measured. DSS treatment resulted in significant increases in colon adenoma number (P < 0.001, n > 5) and burden in Apcmin/+ (P < 0.01, n > 5) and Dclk1Cre/+;Apcfl/fl (P < 0.02, n > 5) mice. There was no effect on adenomas following treatment with PP in combination with ABT263. Adenoma number and burden were reduced with PP+sulindac treatment in Dclk1Cre/+;Apcfl/fl mice (P < 0.01, n > 17) and in Apcmin/+ mice (P < 0.001, n > 7) treated with sulindac or PP+sulindac with no detectable toxicity. PP treatment of Apcmin/+ mice increased the frequency of CD3+ cells in the adenomas. The combination of Wnt pathway inhibition with sulindac was more effective in Dclk1Cre/+;Apcfl/fl mice and provides an opportunity for killing Apc-mutant colon adenoma cells, indicating a strategy for both colorectal cancer prevention and potential new treatments for patients with advanced colorectal cancer. Outcomes from the results of this study may be translatable to the clinic for management of FAP and other patients with a high risk of developing colorectal cancer. Significance: Colorectal cancer is one of the most common cancers worldwide with limited therapeutic options. APC and other Wnt signaling mutations occur in the majority of colorectal cancers but there are currently no Wnt inhibitors in the clinic. The combination of Wnt pathway inhibition with sulindac provides an opportunity for killing Apc-mutant colon adenoma cells and suggests a strategy for colorectal cancer prevention and new treatments for patients with advanced colorectal cancer.

With the advent of faster magic-angle spinning (MAS) and higher magnetic fields, the resolution of biomolecular solid-state nuclear magnetic resonance (NMR) spectra has been continuously increasing. As a direct consequence, the always narrower spectral lines, especially in proton-detected spectroscopy, are also becoming more sensitive to temporal instabilities of the magnetic field in the sample volume. Field drifts in the order of tenths of parts per million occur after probe insertion or temperature change, during cryogen refill, or are intrinsic to the superconducting high-field magnets, particularly in the months after charging. As an alternative to a field–frequency lock based on deuterium solvent resonance rarely available for solid-state NMR, we present a strategy to compensate non-linear field drifts using simultaneous acquisition of a frequency reference (SAFR). It is based on the acquisition of an auxiliary 1D spectrum in each scan of the experiment. Typically, a small-flip-angle pulse is added at the beginning of the pulse sequence. Based on the frequency of the maximum of the solvent signal, the field evolution in time is reconstructed and used to correct the raw data after acquisition, thereby acting in its principle as a digital lock system. The general applicability of our approach is demonstrated on 2D and 3D protein spectra during various situations with a non-linear field drift. SAFR with small-flip-angle pulses causes no significant loss in sensitivity or increase in experimental time in protein spectroscopy. The correction leads to the possibility of recording high-quality spectra in a typical biomolecular experiment even during non-linear field changes in the order of 0.1 ppm h−1 without the need for hardware solutions, such as stabilizing the temperature of the magnet bore. The improvement of linewidths and peak shapes turns out to be especially important for 1H-detected spectra under fast MAS, but the method is suitable for the detection of carbon or other nuclei as well., Magnetic Resonance, 3 (1), ISSN:2699-0016

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignant disease with a 5-year survival rate of

A new heteronuclear decoupling sequence for solid-state NMR and magic angle spinning faster than 60 kHz was recently introduced [Simion et al., J. Chem. Phys. 157, 014202 (2022)]. It was dubbed ROtor-Synchronized Phase-Alternated Cycles (ROSPAC), and it offers robustness for a large range of chemical shifts and low radio-frequency (RF) powers and is almost independent of the radio-frequency power. Here, we theoretically explore the robustness of the ROSPAC sequence toward 1H offset and RF field inhomogeneities, as well as the spacing effect of the π pulses on the decoupling efficiency. We use a generalized theoretical framework based on the Floquet theory to assess these parameters. The optimum decoupling conditions, where the magnitude of the second-order cross-terms and first-order resonance conditions are small, were identified., The Journal of Chemical Physics, 158 (15), ISSN:0021-9606, ISSN:1089-7690

Software complexity of vehicles is constantly growing especially with additional autonomous driving features being introduced. This increases the risk for bugs in the system, when the car is delivered. According to a car manufacturer, more than 90% of availability problems corresponding to Electronic Control Unit (ECU) functionality are either caused by software bugs or they can be resolved by applying software updates to overcome hardware issues. The main concern are sporadic errors which are not caught during the development phase since their trigger condition is too unlikely to occur or is not covered by the tests. For such systems, there is a need of safe and secure infield diagnosis. In this paper we present a tool software architecture with remote access, which facilitates standard read/write access, an efficient channel interface for communication and file I/O, and continuous trace. This enables the remote access of latest automotive Microcontroller Units (MCUs)’ trace systems, which provide non-intrusive system observation without compromising safety, security, or real-time performance. The tool access architecture is designed such that the physical interface is agnostic for the tool. A target can be connected with any standard tool interface as well as with Ethernet. With today’s increased silicon performance, Ethernet can be a viable option as tool interface, from development to the field. The implementation includes an agent firmware that can either run on an application core or as a sand-boxed sub-task of the security core. With the next generation vehicles’ E/E architectures moving towards an Ethernet backbone, this gives developers an option to include remote access to target systems without additional tool hardware.

IL11 is a member of the IL6 family of cytokines and signals through its cognate receptor subunits, IL11RA and glycoprotein 130 (GP130), to elicit biological responses via the JAK/STAT signaling pathway. IL11 contributes to cancer progression by promoting the survival and proliferation of cancer cells, but the potential immunomodulatory properties of IL11 signaling during tumor development have thus far remained unexplored. Here, we have characterized a role for IL11 in regulating CD4+ T cell–mediated antitumor responses. Absence of IL11 signaling impaired tumor growth in a sporadic mouse model of colon cancer and syngeneic allograft models of colon cancer. Adoptive bone marrow transfer experiments and in vivo depletion studies demonstrated that the tumor-promoting activity of IL11 was mediated through its suppressive effect on host CD4+ T cells in the tumor microenvironment. Indeed, when compared with Il11ra-proficient CD4+ T cells associated with MC38 tumors, their Il11ra-deficient counterparts displayed elevated expression of mRNA encoding the antitumor mediators IFNγ and TNFα. Likewise, IL11 potently suppressed the production of proinflammatory cytokines (IFNγ, TNFα, IL6, and IL12p70) by CD4+ T cells in vitro, which we corroborated by RNAscope analysis of human colorectal cancers, where IL11RAhigh tumors showed less IFNG and CD4 expression than IL11RAlow tumors. Therefore, our results ascribe a tumor cell–extrinsic immunomodulatory role to IL11 during colon cancer development that could be amenable to an anticytokine-based therapy.See related Spotlight by van der Burg, p. 724.

Increased HCK activity in BMDMs supports tumor cell invasion in vitro.

Adenomatous polyposis coli (APC) truncations occur in many colorectal cancers and are often associated with immune infiltration. The aim of this study was to determine whether a combination of Wnt inhibition with anti-inflammatory (sulindac) and/or proapototic (ABT263) drugs can reduce colon adenomas. Apcmin/+ and doublecortin-like kinase 1 (Dclk1)Cre/+;Apcfl/fl mice were exposed to dextran sulphate sodium (DSS) in their drinking water to promote the formation of colon adenomas. Mice were then treated with either a Wnt-signaling antagonist pyrvinium pamoate (PP), an anti-inflammatory agent sulindac or proapoptotic compound ABT263 or a combination of PP+ABT263, or PP+sulindac. Colon adenoma frequency, size, and T-cell abundance were measured. DSS treatment resulted in significant increases in colon adenoma number (P < 0.001, n > 5) and burden in Apcmin/+ (P < 0.01, n > 5) and Dclk1Cre/+;Apcfl/fl (P < 0.02, n > 5) mice. There was no effect on adenomas following treatment with PP in combination with ABT263. Adenoma number and burden were reduced with PP+sulindac treatment in Dclk1Cre/+;Apcfl/fl mice (P < 0.01, n > 17) and in Apcmin/+ mice (P < 0.001, n > 7) treated with sulindac or PP+sulindac with no detectable toxicity. PP treatment of Apcmin/+ mice increased the frequency of CD3+ cells in the adenomas. The combination of Wnt pathway inhibition with sulindac was more effective in Dclk1Cre/+;Apcfl/fl mice and provides an opportunity for killing Apc-mutant colon adenoma cells, indicating a strategy for both colorectal cancer prevention and potential new treatments for patients with advanced colorectal cancer. Outcomes from the results of this study may be translatable to the clinic for management of FAP and other patients with a high risk of developing colorectal cancer.Significance:Colorectal cancer is one of the most common cancers worldwide with limited therapeutic options. APC and other Wnt signaling mutations occur in the majority of colorectal cancers but there are currently no Wnt inhibitors in the clinic. The combination of Wnt pathway inhibition with sulindac provides an opportunity for killing Apc-mutant colon adenoma cells and suggests a strategy for colorectal cancer prevention and new treatments for patients with advanced colorectal cancer.

Adenoma tumour development in Apcmin/+ and Dclk1Cre/+;Apcfl/fl mice

Persistent activation of the latent transcription factor STAT3 is observed in gastric tumor epithelial and immune cells and is associated with a poor patient prognosis. Although targeting STAT3-activating upstream kinases offers therapeutically viable targets with limited specificity, direct inhibition of STAT3 remains challenging. Here we provide functional evidence that myeloid-specific hematopoietic cell kinase (HCK) activity can drive STAT3-dependent epithelial tumor growth in mice and is associated with alternative macrophage activation alongside matrix remodeling and tumor cell invasion. Accordingly, genetic reduction of HCK expression in bone marrow–derived cells or systemic pharmacologic inhibition of HCK activity suppresses alternative macrophage polarization and epithelial STAT3 activation, and impairs tumor growth. These data validate HCK as a molecular target for the treatment of human solid tumors harboring excessive STAT3 activity.

Constitutive HCK activity in bone marrow-derived cells enhances STAT3 activation in tumors and tumor-associated macrophages.

Increased expression of genes associated with alternative macrophage polarization confers a poorer overall survival in human intestinal-type gastric cancer patients.

Supplementary Figure 1. Gating strategy and representative flow cytometric plots of MC38 tumors from WT and Il11ra-/- hosts. Supplementary Figure 2. IL-11 has no effect on tumor-infiltrating immune cells in MC38 and CT26 tumors from WT and Il11ra-/- hosts. Supplementary Figure 3. IL-11 has no effect on PMA/Iono-induced IFNγ and TNFα production by IL-11RA-deficient CD4+ T cells. Supplementary Figure 4. Single cell profiling of IL11RA and IFNG expression in intra-tumoral CD4+ T cells from colon cancer patients.

Supplementary Table 1

Histology and immunohistochemical analysis of beta-catenin in adenomas from treated Dclk1Cre/+;Apcfl/fl mice.

Excessive HCK activity in bone marrow-derived cells promotes STAT3-dependent gastric tumor development and invasion.

Xgal and DCLK1 staining of colon crypts from DCLK1-CreERT2 BAC transgenic mice

Fig S2. MC38 tumor growth is enhanced in St2-deficient hosts.

Supplementary Figure 3

Supplementary Table 2

Supplementary Tables 1-11 includes GISTIC results, clinical characteristics, details on cell lines and MACROD2 deletions, pathogenicity predictions, metaphase chromosomes anomalies, multivariate analyses and primer sequences.

Supplementary Figures 1-23 including results for fine mapping of MACROD2 deletions, xenograft assay, immunofluoresence, comet assays, western blots, PARP1 activity assays, clonogenic assays, apoptosis assays, metaphase spreads and chromosome missegregation quantification.

Supplementary Table 1

Fig S3. St2 is not expressed by Lgr5+ stem cells within the colon.

Supplementary Table 3

Supplementary figure 3 shows combining trametinib with different classes of HDAC inhibitors in COLO 201 cells

Fig S1. IL-33 and St2 protein expression in sporadic colon cancer.

The EGFR/RAS/MEK/ERK signaling pathway (ERK/MAPK) is hyperactivated in most colorectal cancers. A current limitation of inhibitors of this pathway is that they primarily induce cytostatic effects in colorectal cancer cells. Nevertheless, these drugs do induce expression of proapoptotic factors, suggesting they may prime colorectal cancer cells to undergo apoptosis. As histone deacetylase inhibitors (HDACis) induce expression of multiple proapoptotic proteins, we examined whether they could synergize with ERK/MAPK inhibitors to trigger colorectal cancer cell apoptosis. Combined MEK/ERK and HDAC inhibition synergistically induced apoptosis in colorectal cancer cell lines and patient-derived tumor organoids in vitro, and attenuated Apc-initiated adenoma formation in vivo. Mechanistically, combined MAPK/HDAC inhibition enhanced expression of the BH3-only proapoptotic proteins BIM and BMF, and their knockdown significantly attenuated MAPK/HDAC inhibitor–induced apoptosis. Importantly, we demonstrate that the paradigm of combined MAPK/HDAC inhibitor treatment to induce apoptosis can be tailored to specific MAPK genotypes in colorectal cancers, by combining an HDAC inhibitor with either an EGFR, KRASG12C or BRAFV600 inhibitor in KRAS/BRAFWT; KRASG12C, BRAFV600E colorectal cancer cell lines, respectively. These findings identify a series of ERK/MAPK genotype-tailored treatment strategies that can readily undergo clinical testing for the treatment of colorectal cancer.

Supplementary Figure 4

Fig S5. Loss of IL-33 signalling does not affect the recruitment of macrophages, mast cells and total T cells to the colon tumors.

Aberrant activation of the latent transcription factor STAT3 and its downstream targets is a common feature of epithelial-derived human cancers, including those of the gastrointestinal tract. Mouse models of gastrointestinal malignancy implicate Stat3 as a key mediator of inflammatory-driven tumorigenesis, in which its cytokine/gp130/Janus kinase (Jak)–dependent activation provides a functional link through which the microenvironment sustains tumor promotion. Although therapeutic targeting of STAT3 is highly desirable, such molecules are not available for immediate clinical assessment. Here, we investigated whether the small-molecule Jak1/2 inhibitor AZD1480 confers therapeutic benefits in two mouse models of inflammation-associated gastrointestinal cancer, which are strictly dependent of excessive Stat3 activation. We confirm genetically that Cre-mediated, tumor cell–specific reduction of Stat3 expression arrests the growth of intestinal-type gastric tumors in gp130F/F mice. We find that systemic administration of AZD1480 readily replicates this effect, which is associated with reduced Stat3 activation and correlates with diminished tumor cell proliferation and increased apoptosis. Likewise, AZD1480 therapy also conferred a cytostatic effect on established tumors in a colitis-associated colon cancer model in wild-type mice. As predicted from our genetic observations in gp130F/F mice, the therapeutic effect of AZD1480 remains fully reversible upon cessation of compound administration. Collectively, our results provide the first evidence that pharmacologic targeting of excessively activated wild-type Jak kinases affords therapeutic suppression of inflammation-associated gastrointestinal cancers progression in vivo. Mol Cancer Ther; 13(2); 468–74. ©2014 AACR.

Supplementary Figure 6

Fig S4. St2 is expressed by colon-derived mesenchymal cells.

Supplementary figure 4 shows phosphorylated ERK staining in an adenoma from a ACDX2 mouse

Supplementary figure 2 shows the effect of trametinib plus panobinostat on colony formation in HCT 116 cells

Supplementary Figure 7

PDF file - 141K, Table S1. Primers used for quantitative PCR analysis. Table S2. Antibodies (all diluted 1:1000) used for immunohistochemistry and Western Blot analysis.

Supplementary figure 5 shows mouse weight during treatment in two of the mouse studies

Supplementary Figure 5

Supplementary Figure 1

Fig S6. St2-deficiency did not alter Treg frequency in untreated naïve colons.

PDF file - 1978K, Supplementary Figure 1: The gpA33 locus confers expression of tamoxifen-activatable CreERT2 transgene to the metaplastic epithelium of gastric tumors of gp130F/F mice. Supplementary Figure 2: AZD1480-treatment reduces gastric tumour burden in gp130F/F mice. Supplementary Figure 3: Immunohistochemical analysis of gastric tumors from AZD1480-treated gp130F/F mice. Supplementary Figure 4: Systemic AZD1480 administration reduces established colitis-associated colon cancer burden in wild-type mice.

Supplementary table 1

Suppl Table 1. Oligonucleotide Sequences

Supplementary Methods for generation of CRISPR and stable cell lines, MACROD2 mutation detection, QRT-PCR, phenotyping of MACROD2/APCmin mice, IHC, WB, TCF reporter assay, RNAseq analysis, generation of MACROD2 antibody, clonogenic assay, PARP1 activity assay, apoptosis assays, IF and FACS analysis.