Continuous Floquet theory in solid-state NMR.

This article presents the application of continuous Floquet theory in solid-state nuclear magnetic resonance (NMR). Continuous Floquet theory extends the traditional Floquet theory to non-continuous Hamiltonians, enabling the description of observable effects not fully captured by the traditional Floquet theory due to its requirement for a periodic Hamiltonian. We present closed-form expressions for computing first- and second-order effective Hamiltonians, streamlining integration with the traditional Floquet theory and facilitating application in NMR experiments featuring multiple modulation frequencies. Subsequently, we show examples of the practical application of continuous Floquet theory by investigating several solid-state NMR experiments. These examples illustrate the importance of the duration of the pulse scheme regarding the width of the resonance conditions and the near-resonance behavior.

Pulsed dynamic nuclear polarization: a comprehensive Floquet description.

Dynamic nuclear polarization (DNP) experiments using microwave (mw) pulse sequences are one approach to transfer the larger polarization on the electron spin to nuclear spins of interest. How the result of such experiments depends on the external magnetic field and the excitation power is part of an ongoing debate and of paramount importance for applications that require high chemical-shift resolution. To date numerical simulations using operator-based Floquet theory have been used to predict and explain experimental data. However, such numerical simulations provide only limited insight into parameters relevant for efficient polarization transfer, such as transition amplitudes or resonance offsets. Here we present an alternative method to describe pulsed DNP experiments by using matrix-based Floquet theory. This approach leads to analytical expressions for the transition amplitudes and resonance offsets. We validate the method by comparing computations by these analytical expressions to their numerical counterparts and to experimental results for the XiX, TOP and TPPM DNP sequences. Our results explain the experimental data and are in very good agreement with the numerical simulations. The analytical expressions allow for the discussion of the scaling behaviour of pulsed DNP experiments with respect to the external magnetic field. We find that the transition amplitudes scale inversely with the external magnetic field.

Combination of bazedoxifene with chemotherapy and SMAC-mimetics for the treatment of colorectal cancer.

Excessive STAT3 signalling via gp130, the shared receptor subunit for IL-6 and IL-11, contributes to disease progression and poor survival outcomes in patients with colorectal cancer. Here, we provide evidence that bazedoxifene inhibits tumour growth via direct interaction with the gp130 receptor to suppress IL-6 and IL-11-mediated STAT3 signalling. Additionally, bazedoxifene combined with chemotherapy synergistically reduced cell proliferation and induced apoptosis in patient-derived colon cancer organoids. We elucidated that the primary mechanism of anti-tumour activity conferred by bazedoxifene treatment occurs via pro-apoptotic responses in tumour cells. Co-treatment with bazedoxifene and the SMAC-mimetics, LCL161 or Birinapant, that target the IAP family of proteins, demonstrated increased apoptosis and reduced proliferation in colorectal cancer cells. Our findings provide evidence that bazedoxifene treatment could be combined with SMAC-mimetics and chemotherapy to enhance tumour cell apoptosis in colorectal cancer, where gp130 receptor signalling promotes tumour growth and progression.

Orthotopic MC-38 Allograft as a Robust Preclinical Model of Colorectal Carcinoma.

Colorectal cancer (CRC) is a significant global health concern, requiring effective preclinical models for studying its development and testing therapies. Mouse models have been used, including spontaneous tumors, carcinogen exposure, and tumor cell implantation as xenografts or at orthotopic sites. Here, we describe an orthotopic preclinical model of CRC, which provides a valuable tool for studying tumor growth and the tumor microenvironment, offering a more accurate representation of human CRC compared to xenograft models.

Screening for Primary Aldosteronism by Mass Spectrometry Versus Immunoassay Measurements of Aldosterone: A Prospective Within-Patient Study.

BACKGROUND: Measurements of aldosterone by mass spectrometry are more accurate and less prone to interferences than immunoassay measurements, and may produce a more accurate aldosterone:renin ratio (ARR) when screening for primary aldosteronism (PA). METHODS: Differences in diagnostic performance of the ARR using mass spectrometry vs immunoassay measurements of aldosterone were examined in 710 patients screened for PA. PA was confirmed in 153 patients and excluded in 451 others. Disease classifications were not achieved in 106 patients. Areas under receiver-operating characteristic curves (AUROC) and other measures were used to compare diagnostic performance. RESULTS: Mass spectrometry-based measurements yielded lower plasma aldosterone concentrations than immunoassay measurements. For the ARR based on immunoassay measurements of aldosterone, AUROCs were slightly lower (P = 0.018) than those using mass spectrometry measurements (0.895 vs 0.906). The cutoff for the ARR to reach a sensitivity of 95% was 30 and 21.5 pmol/mU by respective immunoassay and mass spectrometry-based measurements, which corresponded to specificities of 57% for both. With data restricted to patients with unilateral PA, diagnostic sensitivities of 94% with specificities >81% could be achieved at cutoffs of 68 and 52 pmol/mU for respective immunoassay and mass spectrometry measurements. CONCLUSIONS: Mass spectrometry-based measurements of aldosterone for the ARR provide no clear diagnostic advantage over immunoassay-based measurements. Both approaches offer limited diagnostic accuracy for the ARR as a screening test. One solution is to employ the higher cutoffs to triage patients likely to have unilateral PA for further tests and possible adrenalectomy, while using the lower cutoffs to identify others for targeted medical therapy.German Clinical Trials Register ID: DRKS00017084.

Relaxation enhancement by microwave irradiation may limit dynamic nuclear polarization.

Dynamic nuclear polarization enables the hyperpolarization of nuclear spins beyond the thermal-equilibrium Boltzmann distribution. However, it is often unclear why the experimentally measured hyperpolarization is below the theoretically achievable maximum polarization. We report a (near-) resonant relaxation enhancement by microwave (MW) irradiation, leading to a significant increase in the nuclear polarization decay compared to measurements without MW irradiation. For example, the increased nuclear relaxation limits the achievable polarization levels to around 35% instead of hypothetical 60%, measured in the DNP material TEMPO in 1H glassy matrices at 3.3 K and 7 T. Applying rate-equation models to published build-up and decay data indicates that such relaxation enhancement is a common issue in many samples when using different radicals at low sample temperatures and high Boltzmann polarizations of the electrons. Accordingly, quantification and a better understanding of the relaxation processes under MW irradiation might help to design samples and processes towards achieving higher nuclear hyperpolarization levels.

Identification of Serum Biomarkers to Monitor Therapeutic Response in Intestinal-Type Gastric Cancer.

There are a limited number of clinically useful serum biomarkers to predict tumor onset or treatment response in gastric cancer (GC). For this reason, we explored the serum proteome of the gp130Y757F murine model of intestinal-type gastric cancer (IGC). We identified 30 proteins with significantly elevated expression in early gp130Y757F IGC and 12 proteins that were significantly elevated in late gp130Y757F IGC compared to age- and gender-matched wild-type mice. Within these signatures, there was an overlap of 10 proteins commonly elevated in both early- and late-stage disease. These results highlight the potential to identify serum biomarkers of disease stage. Since IGC in the gp130Y757F model can be reversed following therapeutic inhibition of Interleukin (IL)-11, we explored whether the protein signatures we identified could be used to monitor tumor regression. We compared two different therapeutic modalities and found 5 proteins to be uniquely differentially expressed between control animals and animals halfway through treatment, with 10 differentially expressed at the end of treatment. Our findings highlight the potential to identify reliable biomarkers to track IGC tumor regression in response to treatment.

INEPT and CP transfer efficiencies of dynamic systems in MAS solid-state NMR.

Hartmann-Hahn cross polarization and INEPT polarization transfer are the most popular sequences to increase the polarization of low-γ nuclei in magic-angle spinning solid-state NMR. It is well known that the two methods preferentially lead to polarization transfer in different parts of molecules. Cross polarization works best in rigid segments of the molecule while INEPT-based polarization transfer is efficient in highly mobile segments where (nearly) isotropic motion averages out the dipolar couplings. However, there have only been few attempts to define the time scales of motion that are compatible with cross polarization or INEPT transfer in a more quantitative way. We have used simple isotropic jump models in combination with simulations based on the stochastic Liouville equation to elucidate the time scales of motion that allow either cross polarization or INEPT-based polarization transfer. We investigate which motional time scales interfere with one or both polarization-transfer schemes. We have modeled isolated I-S two-spin systems, strongly-coupled I2S three-spin systems and more loosely coupled I-I-S three-spin systems as well as I3S groups. Such fragments can be used as models for typical environments in fully deuterated and back-exchanged molecules (I-S), for fully protonated molecules (I2S and I3S) or situations in between (I-I-S).

Mechanisms of cellular crosstalk in the gastric tumor microenvironment are mediated by YAP1 and STAT3.

Deregulation of the Hippo pathway is a driver for cancer progression and treatment resistance. In the context of gastric cancer, YAP1 is a biomarker for poor patient prognosis. Although genomic tumor profiling provides information of Hippo pathway activation, the present study demonstrates that inhibition of Yap1 activity has anti-tumor effects in gastric tumors driven by oncogenic mutations and inflammatory cytokines. We show that Yap1 is a key regulator of cell metabolism, proliferation, and immune responses in normal and neoplastic gastric epithelium. We propose that the Hippo pathway is targetable across gastric cancer subtypes and its therapeutic benefits are likely to be mediated by both cancer cell-intrinsic and -extrinsic mechanisms.

Tumor macrophage functional heterogeneity can inform the development of novel cancer therapies.

Macrophages represent a key component of the tumor microenvironment (TME) and are largely associated with poor prognosis. Therapeutic targeting of macrophages has historically focused on inhibiting their recruitment or reprogramming their phenotype from a protumor (M2-like) to an antitumor (M1-like) one. Unfortunately, this approach has not provided clinical breakthroughs that have changed practice. Emerging studies utilizing single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics have improved our understanding of the ontogeny, phenotype, and functional plasticity of macrophages. Overlaying the wealth of current information regarding macrophage molecular subtypes and functions has also identified novel therapeutic vulnerabilities that might drive better control of tumor-associated macrophages (TAMs). Here, we discuss the functional profiling of macrophages and provide an update of novel macrophage-targeted therapies in development.

A tuft cell - ILC2 signaling circuit provides therapeutic targets to inhibit gastric metaplasia and tumor development.

Although gastric cancer is a leading cause of cancer-related deaths, systemic treatment strategies remain scarce. Here, we report the pro-tumorigenic properties of the crosstalk between intestinal tuft cells and type 2 innate lymphoid cells (ILC2) that is evolutionarily optimized for epithelial remodeling in response to helminth infection. We demonstrate that tuft cell-derived interleukin 25 (IL25) drives ILC2 activation, inducing the release of IL13 and promoting epithelial tuft cell hyperplasia. While the resulting tuft cell - ILC2 feed-forward circuit promotes gastric metaplasia and tumor formation, genetic depletion of tuft cells or ILC2s, or therapeutic targeting of IL13 or IL25 alleviates these pathologies in mice. In gastric cancer patients, tuft cell and ILC2 gene signatures predict worsening survival in intestinal-type gastric cancer where ~40% of the corresponding cancers show enriched co-existence of tuft cells and ILC2s. Our findings suggest a role for ILC2 and tuft cells, along with their associated cytokine IL13 and IL25 as gatekeepers and enablers of metaplastic transformation and gastric tumorigenesis, thereby providing an opportunity to therapeutically inhibit early-stage gastric cancer through repurposing antibody-mediated therapies.

Modelling and correcting the impact of RF pulses for continuous monitoring of hyperpolarized NMR.

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 (<3 ∘). 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.

TCF-1 limits intraepithelial lymphocyte antitumor immunity in colorectal carcinoma.

Intraepithelial lymphocytes (IELs), including αß and γδ T cells (T-IELs), constantly survey and play a critical role in maintaining the gastrointestinal epithelium. We show that cytotoxic molecules important for defense against cancer were highly expressed by T-IELs in the small intestine. In contrast, abundance of colonic T-IELs was dependent on the microbiome and displayed higher expression of TCF-1/TCF7 and a reduced effector and cytotoxic profile, including low expression of granzymes. Targeted deletion of TCF-1 in γδ T-IELs induced a distinct effector profile and reduced colon tumor formation in mice. In addition, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers (CRCs) compared with normal healthy colon, which strongly correlated with an enhanced γδ T-IEL effector phenotype and improved patient survival. Our work identifies TCF-1 as a colon-specific T-IEL transcriptional regulator that could inform new immunotherapy strategies to treat CRC.

A Mouse Model for the Rapid and Binomial Assessment of Putative WNT/ß-Catenin Signalling Inhibitors.

Specific signalling thresholds of the WNT/ß-catenin pathway affect embryogenesis and tissue homeostasis in the adult, with mutations in this pathway frequently occurring in cancer. Excessive WNT/ß-catenin activity inhibits murine anterior development associated with embryonic lethality and accounts for the driver event in 80% of human colorectal cancers. Uncontrolled WNT/ß-catenin signalling arises primarily from impairment mutation in the tumour suppressor gene APC that otherwise prevents prolonged stabilisation of ß-catenin. Surprisingly, no inhibitor compounds for WNT/ß-catenin signalling have reached clinical use in part owing to the lack of specific in vivo assays that discriminate between on-target activities and dose-limiting toxicities. Here, we present a simple in vivo assay with a binary outcome whereby the administration of candidate compounds to pregnant and phenotypically normal Apcflox/flox mice can rescue in utero death of Apcmin/flox mutant conceptus without subsequent post-mortem assessment of WNT/ß-catenin signalling. Indeed, the phenotypic plasticity of born Apcmin/flox conceptus enables future refinement of our assay to potentially enable dosage finding and cross-compound comparisons. Thus, we show for the first time the suitability of endogenous WNT/ß-catenin signalling during embryonic development to provide an unambiguous and sensitive mammalian in vivo model to assess the efficacy and bioavailability of potential WNT/ß-catenin antagonists.

The effect of methyl group rotation on 1H-1H solid-state NMR spin-diffusion spectra.

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 and explained 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. Analytical calculations and simulations however show that higher-order coherent contributions dominate the experimentally observed negative peaks in our systems. 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 mask the negative contribution to the peaks.

Theoretical description of pulse induced resonances in the homonuclear PIRATE experiment.

Rotor-synchronous π pulses applied to protons (S) enhance homonuclear polarisation transfer between two spins (I) such as 13C or 15N as long as at least a single I-S heteronuclear dipolar-coupling interaction exists. The enhancement is maximum when the chemical-shift difference Δν between two spins equals an integer multiple, n, of the pulse-modulation frequency, which is half the rotor frequency νr. This condition, applied in the Pulse Induced Resonance with Angular dependent Total Enhancement (PIRATE) experiment, can be generalised for any spacing of the pulses k/νr such that Δν=nνr2k . We show, using average Hamiltonian theory (AHT) and Floquet theory, that the resonance conditions promote a second-order recoupling consisting of a cross-term between the homonuclear and heteronuclear dipolar interactions in a three-spin system. The minimum requirement is a coupling between the two I spins and a coupling of one of the I spins to the S spin. The effective Hamiltonian at the resonance conditions contains three-spin operators of the form 2I1±I2∓Sz with a non-zero effective dipolar coupling. Theoretical analysis shows that the effective strength of the resonance conditions decreases with increasing values of k and n. The theory is backed by numerical simulations, and experimental results on fully labelled 13C-glycine demonstrating the efficiency of the different resonance condition for k=1,2 at various spinning frequencies.

The effect of 1H offset and flip-angle on heteronuclear decoupling efficiency in ROSPAC pulsed sequence: A Floquet description.

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.

Functional roles of SRC signaling in pancreatic cancer: Recent insights provide novel therapeutic opportunities.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignant disease with a 5-year survival rate of <10%. Aberrant activation or elevated expression of the tyrosine kinase c-SRC (SRC) is frequently observed in PDAC and is associated with a poor prognosis. Preclinical studies have revealed a multifaceted role for SRC activation in PDAC, including promoting chronic inflammation, tumor cell proliferation and survival, cancer cell stemness, desmoplasia, hypoxia, angiogenesis, invasion, metastasis, and drug resistance. Strategies to inhibit SRC signaling include suppressing its catalytic activity, inhibiting protein stability, or by interfering with signaling components of the SRC signaling pathway including suppressing protein interactions of SRC. In this review, we discuss the molecular and immunological mechanisms by which aberrant SRC activity promotes PDAC tumorigenesis. We also provide a comprehensive update of SRC inhibitors in the clinic, and discuss the clinical challenges associated with targeting SRC in pancreatic cancer.

Residual proton line width under refocused frequency-switched Lee-Goldburg decoupling in MAS NMR.

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.

Structure-Guided Prediction of the Functional Impact of DCLK1 Mutations on Tumorigenesis.

Doublecortin-like kinase 1 (DCLK1) is a functional serine/threonine (S/T)-kinase and a member of the doublecortin family of proteins which are characterized by their ability to bind to microtubules (MTs). DCLK1 is a proposed cancer driver gene, and its upregulation is associated with poor overall survival in several solid cancer types. However, how DCLK1 associates with MTs and how its kinase function contributes to pro-tumorigenic processes is poorly understood. This review builds on structural models to propose not only the specific functions of the domains but also attempts to predict the impact of individual somatic missense mutations on DCLK1 functions. Somatic missense mutations in DCLK1 are most frequently located within the N-terminal MT binding region and likely impact on the ability of DCLK1 to bind to αß-tubulin and to polymerize and stabilize MTs. Moreover, the MT binding affinity of DCLK1 is negatively regulated by its auto-phosphorylation, and therefore mutations that affect kinase activity are predicted to indirectly alter MT dynamics. The emerging picture portrays DCLK1 as an MT-associated protein whose interactions with tubulin heterodimers and MTs are tightly controlled processes which, when disrupted, may confer pro-tumorigenic properties.