The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of tra... more The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5′-aza-2′-deoxycytidine’ treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not reg...
ABSTRACT Spin-diffusion type polarization transfer among heteronuclei, e.g., C-13 or N-15, plays ... more ABSTRACT Spin-diffusion type polarization transfer among heteronuclei, e.g., C-13 or N-15, plays an important role in many solid-state NMR experiments for structure determination of proteins. In such experiments, chemical-shift compensation is provided by the proton bath ('proton-driven') which can be improved by radio-frequency irradiation of the protons. Here, we address the problem that the polarization-transfer rates depend not only on the intermolecular distance but also on the chemical-shift difference of the two spins. We introduce rf-modulation schemes that allow eliminating the chemical-shift dependence to a large extent over a predefined range. At the same time, the rate constants are maximized. (c) 2014 Elsevier B.V. All rights reserved.
The cells of the intestinal epithelium provide a selectively permeable barrier between the extern... more The cells of the intestinal epithelium provide a selectively permeable barrier between the external environment and internal tissues. The integrity of this barrier is maintained by tight junctions, specialised cell-cell contacts that permit the absorption of water and nutrients while excluding microbes, toxins and dietary antigens. Impairment of intestinal barrier function contributes to multiple gastrointestinal disorders, including food-hypersensitivity, inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Glycoprotein A33 (GPA33) is an intestinal epithelium-specific cell surface marker and member of the CTX group of transmembrane proteins. Roles in cell-cell adhesion have been demonstrated for multiple CTX family members, suggesting a similar function for GPA33 within the gastrointestinal tract. To test a potential requirement for GPA33 in intestinal barrier function, we generated Gpa33(-/-) mice and subjected them to experimental regimens designed to produce food hypersensitivity, colitis and CAC. Gpa33(-/-) mice exhibit impaired intestinal barrier function. This was shown by elevated steady-state immunosurveillance in the colonic mucosa and leakiness to oral TRITC-labelled dextran after short-term exposure to dextran sodium sulphate (DSS) to injure the intestinal epithelium. Gpa33(-/-) mice also exhibit rapid onset and reduced resolution of DSS-induced colitis and a striking increase in the number of colitis-associated tumours produced by treatment with the colon-specific mutagen azoxymethane (AOM) followed by two cycles of DSS. In contrast, Gpa33(-/-) mice treated with AOM alone show no increase in sporadic tumour formation, indicating that their increased tumour susceptibility is dependent on inflammatory stimuli. Finally, Gpa33(-/-) mice display hypersensitivity to food allergens, a common co-morbidity in human patients with IBD. We propose that Gpa33(-/-) mice provide a valuable model to study the mechanisms linking intestinal permeability and multiple inflammatory pathologies. Moreover, this model could facilitate pre-clinical studies aimed at identifying drugs that restore barrier function.
The mammalian adult small intestinal epithelium is a rapidly self-renewing tissue that is maintai... more The mammalian adult small intestinal epithelium is a rapidly self-renewing tissue that is maintained by a pool of cycling stem cells intermingled with Paneth cells at the base of crypts. These crypt base stem cells exclusively express Lgr5 and require Wnt3 or, in its absence, Wnt2b. However, the Frizzled (Fzd) receptor that transmits these Wnt signals is unknown. We determined the expression profile of Fzd receptors in Lgr5(+) stem cells, their immediate daughter cells, and Paneth cells. Here we show Fzd7 is enriched in Lgr5(+) stem cells and binds Wnt3 and Wnt2b. Conditional deletion of the Fzd7 gene in adult intestinal epithelium leads to stem cell loss in vivo and organoid death in vitro. Crypts of conventional Fzd7 knockout mice show decreased basal Wnt signaling and impaired capacity to regenerate the epithelium following deleterious insult. These observations indicate that Fzd7 is required for robust Wnt-dependent processes in Lgr5(+) intestinal stem cells.
We show a theoretical framework, based on triple-mode Floquet theory, to analyze recoupling seque... more We show a theoretical framework, based on triple-mode Floquet theory, to analyze recoupling sequences derived from symmetry-based pulse sequences, which have a non-vanishing effective field and are not rotor synchronized. We analyze the properties of one such sequence, a homonuclear double-quantum recoupling sequence derived from the C72 (1) sequence. The new asynchronous sequence outperforms the rotor-synchronized version for spin pairs with small dipolar couplings in the presence of large chemical-shift anisotropy. The resonance condition of the new sequence is analyzed using triple-mode Floquet theory. Analytical calculations of second-order effective Hamiltonian are performed to compare the efficiency in suppressing second-order cross terms. Experiments and numerical simulations are shown to corroborate the results of the theoretical analysis.
The study of the structure and dynamics of biological systems by various experimental techniques ... more The study of the structure and dynamics of biological systems by various experimental techniques forms the basis of understanding biological processes including enzyme mechanisms, cellular energetics, and molecular recognition. Much of the structural information acquired so far is based on either X-ray diffraction or solution-state NMR . The application of these two techniques requires certain physical characteristics of the molecules studied. X-ray diffraction relies on the formation of crystals as an essential prerequisite to structural studies, while solution-state NMR relies on the solubility of the molecule in a suitable solvent and requires rapid tumbling of the molecules in solution, effectively limiting the size of the systems studied to approximately 100 kDa presently. Although the methods mentioned above have proved invaluable in the study of many systems, these limitations have hindered the studies of entire classes of biological molecules including integral membrane proteins, protein aggregates, and fibrous proteins. In such cases the physical nature of the molecules studied has often precluded successful crystallization, whilst the relatively large size or insufficient solubility of many of these complexes has hindered the application of solution-state NMR.
Zero-quantum coherence generation and reconversion in magic-angle spinning solid-state NMR is ana... more Zero-quantum coherence generation and reconversion in magic-angle spinning solid-state NMR is analyzed. Two methods are discussed based on implementations using symmetry-based pulse sequences that utilize either isotropic J couplings or dipolar couplings. In either case, the decoupling of abundant proton spins plays a crucial role for the efficiency of the zero-quantum generation. We present optimized sequences for measuring zero-quantum single-quantum correlation spectra in solids, achieving an efficiency of 50% in ubiquitin. The advantages and disadvantages of zero-quantum single-quantum over single-quantum single-quantum correlation spectroscopy are explored, and similarities and differences with double-quantum single-quantum correlation spectroscopy are discussed. Finally, possible application of zero-quantum single-quantum experiments to polypeptides, where it can lead to better spectral resolution is investigated using ubiquitin, where we find high efficiency and high selectivity, but also increased line widths in the MQ dimension.
We describe a more broad-banded version of the DREAM double-quantum dipolar-recoupling sequence, ... more We describe a more broad-banded version of the DREAM double-quantum dipolar-recoupling sequence, which is devised by superimposing a phase-alternating RF irradiation scheme, that is, XiX pulses, on top of the original DREAM sequence. We call this sequence XiX(CW) DREAM. The recoupling conditions and the corresponding first-order effective Hamiltonian are analyzed using triple-mode Floquet theory. The performance of the XiX(CW) DREAM sequence is compared to the original DREAM sequence by numerical simulations and experiments on small model substances and the model protein ubiquitin. The results confirm that XiX(CW) DREAM shows a wider recoupling bandwidth compared to that of DREAM, therefore making the choice for the position of the carrier frequency less critical.
The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of tra... more The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5′-aza-2′-deoxycytidine’ treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not reg...
ABSTRACT Spin-diffusion type polarization transfer among heteronuclei, e.g., C-13 or N-15, plays ... more ABSTRACT Spin-diffusion type polarization transfer among heteronuclei, e.g., C-13 or N-15, plays an important role in many solid-state NMR experiments for structure determination of proteins. In such experiments, chemical-shift compensation is provided by the proton bath ('proton-driven') which can be improved by radio-frequency irradiation of the protons. Here, we address the problem that the polarization-transfer rates depend not only on the intermolecular distance but also on the chemical-shift difference of the two spins. We introduce rf-modulation schemes that allow eliminating the chemical-shift dependence to a large extent over a predefined range. At the same time, the rate constants are maximized. (c) 2014 Elsevier B.V. All rights reserved.
The cells of the intestinal epithelium provide a selectively permeable barrier between the extern... more The cells of the intestinal epithelium provide a selectively permeable barrier between the external environment and internal tissues. The integrity of this barrier is maintained by tight junctions, specialised cell-cell contacts that permit the absorption of water and nutrients while excluding microbes, toxins and dietary antigens. Impairment of intestinal barrier function contributes to multiple gastrointestinal disorders, including food-hypersensitivity, inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Glycoprotein A33 (GPA33) is an intestinal epithelium-specific cell surface marker and member of the CTX group of transmembrane proteins. Roles in cell-cell adhesion have been demonstrated for multiple CTX family members, suggesting a similar function for GPA33 within the gastrointestinal tract. To test a potential requirement for GPA33 in intestinal barrier function, we generated Gpa33(-/-) mice and subjected them to experimental regimens designed to produce food hypersensitivity, colitis and CAC. Gpa33(-/-) mice exhibit impaired intestinal barrier function. This was shown by elevated steady-state immunosurveillance in the colonic mucosa and leakiness to oral TRITC-labelled dextran after short-term exposure to dextran sodium sulphate (DSS) to injure the intestinal epithelium. Gpa33(-/-) mice also exhibit rapid onset and reduced resolution of DSS-induced colitis and a striking increase in the number of colitis-associated tumours produced by treatment with the colon-specific mutagen azoxymethane (AOM) followed by two cycles of DSS. In contrast, Gpa33(-/-) mice treated with AOM alone show no increase in sporadic tumour formation, indicating that their increased tumour susceptibility is dependent on inflammatory stimuli. Finally, Gpa33(-/-) mice display hypersensitivity to food allergens, a common co-morbidity in human patients with IBD. We propose that Gpa33(-/-) mice provide a valuable model to study the mechanisms linking intestinal permeability and multiple inflammatory pathologies. Moreover, this model could facilitate pre-clinical studies aimed at identifying drugs that restore barrier function.
The mammalian adult small intestinal epithelium is a rapidly self-renewing tissue that is maintai... more The mammalian adult small intestinal epithelium is a rapidly self-renewing tissue that is maintained by a pool of cycling stem cells intermingled with Paneth cells at the base of crypts. These crypt base stem cells exclusively express Lgr5 and require Wnt3 or, in its absence, Wnt2b. However, the Frizzled (Fzd) receptor that transmits these Wnt signals is unknown. We determined the expression profile of Fzd receptors in Lgr5(+) stem cells, their immediate daughter cells, and Paneth cells. Here we show Fzd7 is enriched in Lgr5(+) stem cells and binds Wnt3 and Wnt2b. Conditional deletion of the Fzd7 gene in adult intestinal epithelium leads to stem cell loss in vivo and organoid death in vitro. Crypts of conventional Fzd7 knockout mice show decreased basal Wnt signaling and impaired capacity to regenerate the epithelium following deleterious insult. These observations indicate that Fzd7 is required for robust Wnt-dependent processes in Lgr5(+) intestinal stem cells.
We show a theoretical framework, based on triple-mode Floquet theory, to analyze recoupling seque... more We show a theoretical framework, based on triple-mode Floquet theory, to analyze recoupling sequences derived from symmetry-based pulse sequences, which have a non-vanishing effective field and are not rotor synchronized. We analyze the properties of one such sequence, a homonuclear double-quantum recoupling sequence derived from the C72 (1) sequence. The new asynchronous sequence outperforms the rotor-synchronized version for spin pairs with small dipolar couplings in the presence of large chemical-shift anisotropy. The resonance condition of the new sequence is analyzed using triple-mode Floquet theory. Analytical calculations of second-order effective Hamiltonian are performed to compare the efficiency in suppressing second-order cross terms. Experiments and numerical simulations are shown to corroborate the results of the theoretical analysis.
The study of the structure and dynamics of biological systems by various experimental techniques ... more The study of the structure and dynamics of biological systems by various experimental techniques forms the basis of understanding biological processes including enzyme mechanisms, cellular energetics, and molecular recognition. Much of the structural information acquired so far is based on either X-ray diffraction or solution-state NMR . The application of these two techniques requires certain physical characteristics of the molecules studied. X-ray diffraction relies on the formation of crystals as an essential prerequisite to structural studies, while solution-state NMR relies on the solubility of the molecule in a suitable solvent and requires rapid tumbling of the molecules in solution, effectively limiting the size of the systems studied to approximately 100 kDa presently. Although the methods mentioned above have proved invaluable in the study of many systems, these limitations have hindered the studies of entire classes of biological molecules including integral membrane proteins, protein aggregates, and fibrous proteins. In such cases the physical nature of the molecules studied has often precluded successful crystallization, whilst the relatively large size or insufficient solubility of many of these complexes has hindered the application of solution-state NMR.
Zero-quantum coherence generation and reconversion in magic-angle spinning solid-state NMR is ana... more Zero-quantum coherence generation and reconversion in magic-angle spinning solid-state NMR is analyzed. Two methods are discussed based on implementations using symmetry-based pulse sequences that utilize either isotropic J couplings or dipolar couplings. In either case, the decoupling of abundant proton spins plays a crucial role for the efficiency of the zero-quantum generation. We present optimized sequences for measuring zero-quantum single-quantum correlation spectra in solids, achieving an efficiency of 50% in ubiquitin. The advantages and disadvantages of zero-quantum single-quantum over single-quantum single-quantum correlation spectroscopy are explored, and similarities and differences with double-quantum single-quantum correlation spectroscopy are discussed. Finally, possible application of zero-quantum single-quantum experiments to polypeptides, where it can lead to better spectral resolution is investigated using ubiquitin, where we find high efficiency and high selectivity, but also increased line widths in the MQ dimension.
We describe a more broad-banded version of the DREAM double-quantum dipolar-recoupling sequence, ... more We describe a more broad-banded version of the DREAM double-quantum dipolar-recoupling sequence, which is devised by superimposing a phase-alternating RF irradiation scheme, that is, XiX pulses, on top of the original DREAM sequence. We call this sequence XiX(CW) DREAM. The recoupling conditions and the corresponding first-order effective Hamiltonian are analyzed using triple-mode Floquet theory. The performance of the XiX(CW) DREAM sequence is compared to the original DREAM sequence by numerical simulations and experiments on small model substances and the model protein ubiquitin. The results confirm that XiX(CW) DREAM shows a wider recoupling bandwidth compared to that of DREAM, therefore making the choice for the position of the carrier frequency less critical.
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Papers by Matthias Ernst