Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines.

Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.

Fecal dysbiosis and inflammation in intestinal-specific Cftr knockout mice on regimens preventing intestinal obstruction.

Chronic intestinal inflammation is a poorly understood manifestation of cystic fibrosis (CF), which may be refractory to ion channel CF transmembrane conductance regulator (CFTR) modulator therapy. People with CF exhibit intestinal dysbiosis, which has the potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia, leukocytes, and other tissues. Here, we investigate the contribution of intestinal epithelium-specific loss of Cftr [iCftr knockout (KO)] to dysbiosis and inflammation in mice treated with either of two antiobstructive dietary regimens necessary to maintain CF mouse models [polyethylene glycol (PEG) laxative or a liquid diet (LiqD)]. Feces collected from iCftr KO mice and their wild-type (WT) sex-matched littermates were used to measure fecal calprotectin to evaluate inflammation and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT mice that consumed either PEG or LiqD. PEG iCftr KO mice did not show a change in α diversity versus WT mice but demonstrated a significant difference in microbial composition (ß diversity) with included increases in the phylum Proteobacteria, the family Peptostreptococcaceae, four genera of Clostridia including C. innocuum, and the mucolytic genus Akkermansia. Fecal microbiome analysis of LiqD-fed iCftr KO mice showed both decreased α diversity and differences in microbial composition with increases in the Proteobacteria family Enterobacteriaceae, Firmicutes families Clostridiaceae and Peptostreptococcaceae, and enrichment of Clostridium perfringens, C. innocuum, C. difficile, mucolytic Ruminococcus gnavus, and reduction of Akkermansia. It was concluded that epithelium-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of pan Cftr KO mice.NEW & NOTEWORTHY Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CF transmembrane conductance regulator (CFTR) that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR [inducible Cftr knockout (KO)] in mice is sufficient to induce intestinal dysbiosis and inflammation. Experiments were performed on mice consuming two dietary regimens routinely used to prevent obstruction in CF mice.

Fecal Dysbiosis and Inflammation in Intestinal-Specific Cftr Knockout Mice on Regimens Preventing Intestinal Obstruction.

Chronic intestinal inflammation is a poorly understood manifestation of Cystic Fibrosis (CF), which may be refractory to ion channel CFTR modulator therapy. People with CF exhibit intestinal dysbiosis which has potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia and in the leukocyte population. Here, we investigate the contribution of intestinal epithelial-specific loss of Cftr (iCftr KO) to dysbiosis and inflammation in mice treated with either of two anti-obstructive dietary regimens necessary to maintain CF mouse models (PEG laxative or a liquid diet, LiqD). Feces collected from iCftr KO mice and their wildtype (WT) sex-matched littermates were used to measure fecal calprotectin and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT samples of mice consuming either PEG or LiqD. PEG iCftr KO mice did not show a change in α-diversity versus WT but demonstrated a significant difference in microbial composition (ß-diversity) with increases in phylum Proteobacteria , family Peptostreptococcaceae , four genera of Clostridia including C. innocuum , and mucolytic genus Akkermansia . Fecal microbiome analysis of LiqD iCftr KO mice showed both decreased α-diversity and differences in microbial composition with increases in Proteobacteria family Enterobacteriaceae , Firmicutes families Clostridiaceae and Peptostreptococcaceae , and enrichment of Clostridium perfringens , C. innocuum , C. difficile , mucolytic Ruminococcus gnavus , and reduction of Akkermansia . It was concluded that epithelial-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of global Cftr KO mice. New and noteworthy: Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CFTR that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR (iCftr KO) in mice is sufficient to induce intestinal dysbiosis and inflammation. Studies were performed on mice consuming either dietary regimen (PEG laxative or liquid diet) routinely used to prevent obstruction in CF mice.

Goblet cell hyperplasia is not epithelial-autonomous in the Cftr knockout intestine.

Goblet cell hyperplasia is an important manifestation of cystic fibrosis (CF) disease in epithelial-lined organs. Explants of CF airway epithelium show normalization of goblet cell numbers; therefore, we hypothesized that small intestinal enteroids from Cftr knockout (KO) mice would not exhibit goblet cell hyperplasia. Toll-like receptors 2 and 4 (Tlr2 and Tlr4) were investigated as markers of inflammation and influence on goblet cell differentiation. Ex vivo studies found goblet cell hyperplasia in Cftr KO jejunum compared with wild-type (WT) mice. IL-13, SAM pointed domain-containing ETS transcription factor (Spdef), Tlr2, and Tlr4 protein expression were increased in Cftr KO intestine relative to WT. In contrast, WT and Cftr KO enteroids did not exhibit differences in basal or IL-13-stimulated goblet cell numbers, or differences in expression of Tlr2, Tlr4, and Spdef. Ileal goblet cell numbers in Cftr KO/Tlr4 KO and Cftr KO/Tlr2 KO mice were not different from Cftr KO mice, but enumeration was confounded by altered mucosal morphology. Treatment with Tlr4 agonist LPS did not affect goblet cell numbers in WT or Cftr KO enteroids, whereas the Tlr2 agonist Pam3Csk4 stimulated goblet cell hyperplasia in both genotypes. Pam3Csk4 stimulation of goblet cell numbers was associated with suppression of Notch1 and Neurog3 expression and upregulated determinants of goblet cell differentiation. We conclude that goblet cell hyperplasia and inflammation of the Cftr KO small intestine are not exhibited by enteroids, indicating that this manifestation of CF intestinal disease is not epithelial-automatous but secondary to the altered CF intestinal environment.NEW & NOTEWORTHY Studies of small intestinal organoids from cystic fibrosis (CF) mice show that goblet cell hyperplasia and increased Toll-like receptor 2/4 expression are not primary manifestations of the CF intestine. Intestinal goblet cell hyperplasia in the CF mice was not strongly altered by genetic ablation of Tlr2 and Tlr 4, but could be induced in both wild-type and CF intestinal organoids by a Tlr2-dependent suppression of Notch signaling.

A comparison of impulse response modification techniques for time reversal with application to crack detection.

Time reversal (TR) focusing used for nonlinear detection of cracks relies on the ability of the TR process to provide spatially localized, high-amplitude excitation. The high amplitude improves the ability to detect nonlinear features that are a signature of the motion of closed cracks. It follows that a higher peak focal amplitude than what can be generated with the traditional TR process will improve the detection capability. Modifying the time-reversed impulse response to increase the amplitude of later arrivals in the impulse response, while maintaining the phase information of all arrivals, increases the overall focal signal amplitude. A variety of existing techniques for increasing amplitude are discussed, and decay compensation TR, a technique wherein amplitude is increased according to the inverse of the amplitude envelope of the impulse response decay, is identified as the best modification technique for nonlinear crack detection. This technique increases the focal signal amplitude with a minor introduction of harmonic content, a drawback in two other methods studied, one-bit TR and clipping TR. A final study employs both decay compensation TR and traditional TR, focusing on a rod with stress corrosion cracking, and compares the merits of each in detecting nonlinearity from cracks in a real system.

Nonlinearity from stress corrosion cracking as a function of chloride exposure time using the time reversed elastic nonlinearity diagnostic.

The Time Reversed Elastic Nonlinearity Diagnostic (TREND) has a long history of successful nondestructive detection of cracks in solids using nonlinear indicators. Recent research implemented TREND to find stress corrosion cracking (SCC) in the heat-affected zone adjacent to welds in stainless steel. SCC development around welds is likely to occur due to the temperature and chemical exposure of steel canisters housing spent nuclear fuel. The ideal SCC detection technique would quantify the size and extent of the SCC, rather than just locating it, as TREND has been used for in the past. The current paper explores TREND's ability to detect an assumed increase in SCC over time using 13 samples exposed to a magnesium chloride (MgCl2) bath for different lengths of time. The samples are then scanned with TREND and nonlinearity is quantified for each scan point and each sample. The results suggest that TREND can be used to not only locate SCC in the heat-affected zone, but also track an increase in nonlinearity, and thereby an increase in damage, in samples exposed to the MgCl2 solution for a longer duration.

Time reversal focusing of high amplitude sound in a reverberation chamber.

Time reversal (TR) is a signal processing technique that can be used for intentional sound focusing. While it has been studied in room acoustics, the application of TR to produce a high amplitude focus of sound in a room has not yet been explored. The purpose of this study is to create a virtual source of spherical waves with TR that are of sufficient intensity to study nonlinear acoustic propagation. A parameterization study of deconvolution, one-bit, clipping, and decay compensation TR methods is performed to optimize high amplitude focusing and temporal signal focus quality. Of all TR methods studied, clipping is shown to produce the highest amplitude focal signal. An experiment utilizing eight horn loudspeakers in a reverberation chamber is done with the clipping TR method. A peak focal amplitude of 9.05 kPa (173.1 dB peak re 20 µPa) is achieved. Results from this experiment indicate that this high amplitude focusing is a nonlinear process.

Time reversal focusing of elastic waves in plates for an educational demonstration.

The purpose of this research is to develop a visual demonstration of time reversal focusing of vibrations in a thin plate. Various plate materials are tested to provide optimal conditions for time reversal focusing. Specifically, the reverberation time in each plate and the vibration coupling efficiency from a shaker to the plate are quantified to illustrate why a given plate provides the best spatially confined focus as well as the highest focal amplitude possible. A single vibration speaker and a scanning laser Doppler vibrometer (SLDV) are used to provide the time reversal focusing. Table salt is sprinkled onto the plate surface to allow visualization of the high amplitude, spatially localized time reversal focus; the salt is thrown upward only at the focal position. Spatial mapping of the vibration focusing on the plate using the SLDV is correlated to the visual salt jumping demonstration. The time reversal focusing is also used to knock over an object when the object is placed at the focal position; some discussion of optimal objects to use for this demonstration are given.

Stretch regulates expression and binding of chymotrypsin-like elastase 1 in the postnatal lung.

Lung stretch is critical for normal lung development and for compensatory lung growth after pneumonectomy (PNX), but the mechanisms by which strain induces matrix remodeling are unclear. Our prior work demonstrated an association of chymotrypsin-like elastase 1 (Cela1) with lung elastin remodeling, and that strain triggered a near-instantaneous elastin-remodeling response. We sought to determine whether stretch regulates Cela1 expression and Cela1 binding to lung elastin. In C57BL/6J mice, Cela1 protein increased 176-fold during lung morphogenesis. Cela1 was covalently bound to serpin peptidase inhibitor, clade A, member 1, resulting in a higher molecular mass in lung homogenate compared to pancreas homogenate. Post-PNX, Cela1 mRNA increased 6-fold, protein 3-fold, and Cela1-positive cells 2-fold. Cela1 was expressed predominantly in alveolar type II cells in the embryonic lung and predominantly in CD90-positive lung fibroblasts postnatally. During compensatory lung growth, Cela1 expression was induced in nonproliferative mesenchymal cells. In ex vivo mouse lung sections, stretch increased Cela1 binding to lung tissue by 46%. Competitive inhibition with soluble elastin completely abrogated this increase. Areas of stretch-induced elastase activity and Cela1 binding colocalized. The stretch-dependent expression and binding kinetics of Cela1 indicate an important role in stretch-dependent remodeling of the peripheral lung during development and regeneration.

Outdoor measurements of spherical acoustic shock decay.

Prior anechoic measurements of a small acetylene-oxygen balloon explosion were used to study spherical weak-shock decay over short ranges [Muhlestein et al., J. Acoust. Soc. Am. 131, 2422-2430 (2012)]. Here, longer-range measurements conducted at the Bonneville Salt Flats with a larger balloon are described. Waveform and spectral characteristics and comparisons of the peak pressure decay with an analytical weak-shock model are presented. Weak shocks persist to at least 305 m, with an amplitude decay that is predicted reasonably well using the model. Deviations are discussed in the context of atmospheric effects and nonlinear ground reflections.

JNK suppresses pulmonary fibroblast elastogenesis during alveolar development.

BACKGROUND: The formation of discrete elastin bands at the tips of secondary alveolar septa is important for normal alveolar development, but the mechanisms regulating the lung elastogenic program are incompletely understood. JNK suppress elastin synthesis in the aorta and is important in a host of developmental processes. We sought to determine whether JNK suppresses pulmonary fibroblast elastogenesis during lung development. METHODS: Alveolar size, elastin content, and mRNA of elastin-associated genes were quantitated in wild type and JNK-deficient mouse lungs, and expression profiles were validated in primary lung fibroblasts. Tropoelastin protein was quantitated by Western blot. Changes in lung JNK activity throughout development were quantitated, and pJNK was localized by confocal imaging and lineage tracing. RESULTS: By morphometry, alveolar diameters were increased by 7% and lung elastin content increased 2-fold in JNK-deficient mouse lungs compared to wild type. By Western blot, tropoelastin protein was increased 5-fold in JNK-deficient lungs. Postnatal day 14 (PND14) lung JNK activity was 11-fold higher and pJNK:JNK ratio 6-fold higher compared to PN 8 week lung. Lung tropoelastin, emilin-1, fibrillin-1, fibulin-5, and lysyl oxidase mRNAs inversely correlated with lung JNK activity during alveolar development. Phosphorylated JNK localized to pulmonary lipofibroblasts. PND14 JNK-deficient mouse lungs contained 7-fold more tropoelastin, 2,000-fold more emilin-1, 800-fold more fibrillin-1, and 60-fold more fibulin-5 than PND14 wild type lungs. Primarily lung fibroblasts from wild type and JNK-deficient mice showed similar differences in elastogenic mRNAs. CONCLUSIONS: JNK suppresses fibroblast elastogenesis during the alveolar stage of lung development.

A study of fragmentation of protonated amides of some acylated amino acids by tandem mass spectrometry: observation of an unusual nitrilium ion.

A tandem mass spectrometric study of a series of secondary amides of acetylglycine and hippuric acid utilizing electrospray ionization (ESI) was conducted. Among the fragment ions observed was an unusual one, which we have determined to be a nitrilium ion having the structure CH3-C≡N⊕-Ph or Ph-C≡N⊕-Ph by loss of the full mass of glycine as a neutral fragment. A mechanism that we propose involves an initial protonation of the oxygen atom at the N-terminus, followed by cyclization to a five-membered imidazolium ring, and its subsequent collapse to the nitrilium ion. This mechanism is supported by extensive isotopic labels and considerable variation of substituents. A similar study of the amides of acyl ß-alanine and acyl γ-aminobutyric acid revealed that the former furnishes the same nitrilium ion, but not the latter. Thus, a six-membered intermediate is also possible and capable of losing the full mass of ß-alanine as a neutral fragment. When the size of the ring is forced to be seven-membered, this pathway is blocked. When this study was expanded to include a variety of N-acylproline amides, the nitrilium ion was observed in 100% abundance only when the acyl group was acetyl. Thus a proline effect (involvement of a strained bicyclic [3.3.0] structure) is being observed.