Mechanisms of resistance to selective RET tyrosine kinase inhibitors in RET fusion-positive non-small-cell lung cancer.

BACKGROUND: Rearranged during transfection (RET) gene fusions are a validated target in non-small-cell lung cancer (NSCLC). RET-selective inhibitors selpercatinib (LOXO-292) and pralsetinib (BLU-667) recently demonstrated favorable antitumor activity and safety profiles in advanced RET fusion-positive NSCLC, and both have received approval by the US Food and Drug Administration for this indication. Insights into mechanisms of resistance to selective RET inhibitors remain limited. PATIENTS AND METHODS: This study was performed at five institutions. Tissue and/or cell-free DNA was obtained from patients with RET fusion-positive NSCLC after treatment with selpercatinib or pralsetinib and assessed by next-generation sequencing (NGS) or MET FISH. RESULTS: We analyzed a total of 23 post-treatment tissue and/or plasma biopsies from 18 RET fusion-positive patients who received an RET-selective inhibitor (selpercatinib, n = 10; pralsetinib, n = 7; pralsetinib followed by selpercatinib, n = 1, with biopsy after each inhibitor). Three cases had paired tissue and plasma samples, of which one also had two serial resistant tissue specimens. The median progression-free survival on RET inhibitors was 6.3 months [95% confidence interval 3.6-10.8 months]. Acquired RET mutations were identified in two cases (10%), both affecting the RET G810 residue in the kinase solvent front. Three resistant cases (15%) harbored acquired MET amplification without concurrent RET resistance mutations, and one specimen had acquired KRAS amplification. No other canonical driver alterations were identified by NGS. Among 16 resistant tumor specimens, none had evidence of squamous or small-cell histologic transformation. CONCLUSIONS: RET solvent front mutations are a recurrent mechanism of RET inhibitor resistance, although they occurred at a relatively low frequency. The majority of resistance to selective RET inhibition may be driven by RET-independent resistance such as acquired MET or KRAS amplification. Next-generation RET inhibitors with potency against RET resistance mutations and combination strategies are needed to effectively overcome resistance in these patients.

First report of hepatobiliary Mycobacterium avium infection developing obstructive jaundice in a patient with neutralizing anti-interferon-gamma autoantibodies.

This study describes a patient who experienced hepatobiliary Mycobacterium avium infection associated with neutralizing anti-interferon gamma (IFN-γ) autoantibodies during treatment for disseminated M. avium disease. Hepatobiliary M. avium infection should be considered in jaundiced patients with neutralizing anti-IFN-γ autoantibodies, including those receiving antimycobacterial therapy for disseminated M. avium disease.

Translucent, hydrophobic, and mechanically tough aerogels constructed from trimethylsilylated chitosan nanofibers.

Cross-linking and trimethylsilylation successfully block off the hydrophilic NH2 and OH groups in chitosan nanofibers to produce a waterproof nanofibrous aerogel while keeping its nanoscale structural homogeneity intact. The unique microstructure of a three-dimensionally entangled nanofiber network exhibiting a combination of translucency, hydrophobicity, and non-brittleness is described.

Topographic effects on adhesive force mapping of stretched DNA molecules by pulsed-force-mode atomic force microscopy.

Adhesive interaction between a tip and a sample surface was examined on a microscopic scale by pulsed-force-mode atomic force microscopy (PFM-AFM). The signal measured by monitoring pull-off force is influenced by various factors such as topography, elasticity, electrostatic charges, and adsorbed water on surfaces. Here, we focus on the topographic effects on the adhesive interaction. To clarify the topographic influence, the adhesive force measurement of a stretched DNA molecule with a smaller radius of curvature than that of a tip was carried out at low relative humidity (RH) with an alkanethiol-modified tip. The experimental conditions such as low RH and the use of the alkanethiol-modified tip were required to minimise the influence of water capillary force on hydrated DNA strands. The hydrophobic modification of a substrate surface was also important to minimise the adsorbed water effect. The DNA molecules were stretched on the substrate surfaces by an immobilisation process called a dynamic molecular combing method. The two-component vapour-phase surface modification with an alkylsilane mixed with a silane derivative containing an amino end group enhanced the DNA adsorption due to the electrostatic interaction. The experimental results for the topographic effects on the adhesive force mapping were reproducible.

Runx2-deficient mice lack mandibular condylar cartilage and have deformed Meckel's cartilage.

Runx2 (runt-related transcription factor 2) deficient mice lacked the mandibular condylar cartilage and the mandibular bone. The anlage of the condylar process consisted of mesenchymal condensation, which expressed Type I collagen mRNA and alkaline phosphatase activity, but not Type II collagen and aggrecan mRNAs. Therefore, the differentiation of the mandibular condylar cartilage stopped at the preosteoblast (skeletoblast) stage. The lateral pterygoid muscle was attached to this anlage, and relatively abundant mesenchymal condensations were also formed at the muscle-attaching sites, e.g. the anlage of the mandibular body, the angular and coronoid processes. Three-dimensional reconstruction models showed that each mesenchymal condensation was connected to one another, and roughly outlined the shape of the mandible. Meckel's cartilage in the Runx2-deficient mice had two ectopic cartilaginous processes to which the digastric and myohyoid muscles were attached. These findings indicate that Runx2 is essential for the formation of the mandibular condylar cartilage, as well as for normal development of Meckel's cartilage and that muscle tissues influence mandible morphology.

Orthodontic treatment of a case of Becker muscular dystrophy.

Becker muscular dystrophy, similar to Duchenne muscular dystrophy, is a X-chromosomal linked anomaly characterized by progressive muscle wasting and weakness. Duchenne-type is known to have severe openbite with a steep mandibular plane, but there are no studies that describe the occlusal and skeletal patterns of the Becker-type. Here, we report the orthodontic treatment of a Becker muscular dystrophy patient. In the correction of his severe skeletal open bite general anesthesia or orthognathic surgery was not an option. Multiloop edgewise archwires were employed for orthodontic treatment. After 3 years and 8 months the open bite was corrected. During the retention period contact between the anterior teeth was maintained 8 months after active treatment despite a marked relapse tendency.

Protein crystallization by using porous glass substrate.

The effects of a commercially available porous glass substrate (Corning Porous Glass No.7930) on the heterogeneous nucleation of proteins [hen egg-white lysozyme (HEWL), thaumatin and apoferritin] have been investigated in order to develop an improved method to facilitate the nucleation of protein crystals. It was found that the porous glass substrate could promote the nucleation at lower supersaturations. The induction time for nucleation decreased, and the crystals obtained from porous glass substrates were larger than those from normal glass substrates. Many pores and channels of 10-100 nm in diameter were observed on the porous glass surface by atomic force microscopy (AFM). It is believed that these pores and channels are crucial for facilitating the nucleation process in this work.

Systematic analysis of supersaturation and lysozyme crystal quality.

A systematic study of the correlation between supersaturation and protein crystal quality was carried out employing atomic force microscopy (AFM) and X-ray crystallography with synchrotron radiation (SR). The surface morphology and growth rates of hen egg-white (HEW) lysozyme crystals soaked in various supersaturated solutions were first investigated by AFM. The results showed that the formation of two-dimensional islands increased as a function of supersaturation. The growth rate (molecule intake speed) also increased as a function of supersaturation. In order to examine the correlation between the surface morphology, growth rate and the crystal quality, X-ray diffraction experiments were performed. It was confirmed that crystals grown at lower supersaturations diffracted better with higher signal-to-noise ratios, including better agreement between symmetry-related reflections. The results strongly suggested that the molecular misorientation at high supersaturation affected the crystal quality.

Effects of microgravity on c-fos gene expression in osteoblast-like MC3T3-E1 cells.

The paper summarizes the data on proliferation and gravity-related gene expression of osteoblasts that were obtained from an experiment conducted under simulated and real microgravity conditions. Simulated microgravity conditions obtained in a clinostat depress proliferation of both osteoblast-like MC3T3-E1 and HeLa carcinoma cells. This depression of proliferation occurs in a collagen gel culture in which the flow of culture medium by rotation may be reduced. Interestingly, MC3T3-E1 cells which are probably one of target cells to microgravity are more sensitive than the HeLa cells. Simulated microgravity inhibited the epidermal growth factor (EGF)-induced c-fos gene expression in the MC3T3-El cells. To examine in detail the effect of real microgravity on the EGF signal transduction cascade in osteoblasts, MC3T3-E1 cells were cultured in the Cell Culture Experiment Module of the sounding rocket TR-1A6. The EGF-induced c-fos expression in cells was depressed under short-term microgravity conditions in the sounding rocket, while the phosphorylation of mitogen-activated protein kinase (MAPK) was not affected compared with the controls grown on the ground. These results suggest that an action site of microgravity in the signal transduction pathway may be downstream of MAPK.

Magnetic resonance imaging in diagnosis of right coronary arteriovenous fistula--a case report.

We describe a patient with a right coronary arteriovenous fistula in whom magnetic resonance imaging (MRI) was useful in establishing the diagnosis. In a 36-year-old woman, T1 spin echo MRI demonstrated a massively dilated coronary arteriovenous fistula connecting the right coronary artery to the right atrium. The cine field echo technique showed a continuous shunt flow within the fistula as documented by the flow void throughout the cardiac cycle. These findings were confirmed by cardiac catheterization. We conclude that MRI is useful not only in detecting a coronary arteriovenous fistula but also in identifying its origin and the drainage site.

An autopsy case of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) with a point mutation of mitochondrial DNA.

A 14-year-old boy with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is reported. He had suffered blepharoptosis and cataracts prior to the stroke-like episodes, and was thus reported in 1984 as having Kearns-Shy (Sayre) syndrome. After his death, an A-to-G mutation of the mitochondrial DNA (mtDNA) at bp 3243 was identified in cardiac muscle and the liver. Neuropathologically, multiple old and recent necrotic foci were observed in the gray and white matter of the cerebrum and cerebellum. These lesions were occasionally observed in areas outside of the distribution of major blood vessels of the brain. In the recent necrotic foci, neural loss and sponginess were observed while some neurons were preserved intact. The latter finding has not been described in MELAS and suggests that metabolic degeneration had occurred in the neurons of this patient. This is the first report of a confirmed 3243 mutation of the mtDNA in an autopsied MELAS case.

Resin glycosides. XV. Simonins I-V, ether-soluble resin glycosides (jalapins) from the roots of Ipomoea batatas (cv. Simon).

Five new ether-soluble resin glycosides (jalapins), simonins I-V, have been isolated from the roots of Ipomoea batatas and characterized on the bases of chemical and spectral data. Simonin I is the first example of resin glycoside with aromatic acid (trans-cinnamic acid) as a component organic acid.

Potential bile acid metabolites. 16. Synthesis of stereoisomeric 3 alpha,6,7,12 alpha-tetrahydroxy-5 beta-cholanoic acids.

New synthetic routes to the four possible stereoisomeric 3 alpha,6,7,12 alpha-tetrahydroxy-5 beta-cholanoic acids (and their methyl esters), one of which (3 alpha,6 alpha 7 beta,12 alpha) is new, and some related compounds are described. In addition, the 5 alpha-epimer of the new acid was obtained. The final products were obtained in high purity for use as reference compounds in the analysis of bile acids in human biologic samples. The results of analysis of the prepared stereoisomers by proton and carbon 13 nuclear magnetic resonance spectroscopies are briefly discussed along with the thin-layer and gas-liquid chromatographic properties.

CCCP activation of the reconstituted NaK-pump.

In the NaK-ATPase proteoliposomes (PLs), the NaK-pump activity, Na+ uptake, and ATP hydrolysis were apparently enhanced by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and other ionophores without ion gradients. These ionophore effects were not cation specific. Without ionophores, the PL's ATPase activity fell to its steady-state value within 3 sec at 15 degrees C. This decrease in activity disappeared in the presence of CCCP. Since CCCP is believed to enhance proton mobility across the lipid bilayer and dissipate membrane potential (Vm), we postulated that a Vm build-up partially inhibits the PLs by changing the conformation of the NaK-pump, and that CCCP eliminated this partial inhibition. Since this activation required extracellular K+ and high ATP concentration in the PLs, CCCP must affect the conversion between the phosphorylated forms of NaK-ATPase (EP); this step has been suggested by Goldschlegger et al. (1987) to be the voltage-sensitive step (J. Physiol. (London) 387:331-355). Although cytoplasmic K+ accelerated the change of ADP- and K(+)-sensitive EP (E*P) to K(+)-sensitive ADP-insensitive EP (E2P), CCCP did not complete with cytoplasmic K+ when cytoplasmic Na+ was saturated. When the PLs were phosphorylated with 20 microM ATP and 20 microM palmitoyl CoA instead of with high concentration of ATP, CCCP increased the E*P content and decreased the ADP-sensitive K(+)-insensitive EP (E1P). The results described above suggest that CCCP affects the E1P to E*P change in the E1P----E*P----E2P conversion and that this reaction step is inhibited by Vm.

Cytoplasmic K+ effects on phosphoenzyme of Na,K-ATPase proteoliposomes and on the Na+-pump activity.

Three phosphorylated reaction intermediates (EP) of Na,K-ATPase, and ADP-sensitive K+-insensitive EP (E1P), an ADP- and K+-sensitive EP (E*P), and a K+-sensitive ADP-insensitive EP (E2P), have been discovered at present. By using Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme, we found in this study that E*P existed even in the presence of K+ on both sides of the PL and that there was a sidedness difference in K+ sites between E*P and E2P. Cytoplasmic K+ (K+cyt) accelerated the conversion of E*P to E2P but did not dephosphorylate the E2P. Although the extracellular K+ accelerated the dephosphorylation of E2P, it did not interact with E*P directly. This K+cyt effect was also verified by the activation of Na+-pump in the Na+-K+ exchange mode. In the presence of K+cyt, both the ATP hydrolysis and Na+ uptake rates of the PL containing K+ inside vesicles increased sigmoidally with the concentrations of ATP and cytoplasmic Na+ (Na+cyt). However, in the absence of K+cyt, these Na+-pump reactions in PL containing K+ inside vesicles had only a hyperbolic curve. These results imply that the E*P to E2P conversion is one of the rate-limiting steps of the Na+-pump in the presence of a high concentration of ATP and that K+cyt may control this reaction step by enhancing the conversion rate of E*P to E2P.

Phosphorylated intermediates of Na,K-ATPase proteoliposomes controlled by bilayer cholesterol. Interaction with cardiac steroid.

Fragmental Na,K-ATPase from the electric eel forms three phosphorylated intermediates (EP) with MgATP and Na+: ADP-sensitive K+-insensitive EP (E1P), ADP- and K+-sensitive EP (E*P), and K+-sensitive ADP-insensitive EP (E2P). The EP composition varied with the Na+ concentration. In the reconstituted Na,K-ATPase proteoliposomes (PL), the EP composition of the inside-out form was controlled not only by the intravesicular (extracellular) Na+ concentration, but also by the temperature and the cholesterol content of the lipid bilayer. When the lipid bilayer of PL contained less than 30 mol % cholesterol, the E*P content did not change significantly while the E2P content increased with an elevation in temperature (3-20 degrees C). In contrast, when the lipid bilayer contains more than 35 mol % cholesterol, the E*P content increased while the E2P content stayed less than 10% under the same temperature change. These observations suggest that a high cholesterol content in the lipid bilayer interferes with the E*P to E2P conversion. This cholesterol effect was reversed by ionophores (monensin, nigericin, and A23187). Therefore, E1P-rich EP, E*P-rich EP, or E2P-rich EP could be obtained in the PL under a constant Na+ concentration by using various concentrations of cholesterol and ionophores. The reaction between the proteoliposomal EPs and digitoxigenin (lipid-soluble cardiac steroid) occurred in a single turnover, thereby avoiding unphysiologically high Na+ concentrations. The increase in the ADP- and K+-insensitive EP, which indicated formation of the digitoxigenin-Na,K-ATPase complex, was equivalent to the decrease in the E*P under six different sets of conditions, without any significant change in the E1P and E2P contents. This result indicated that E*P is the active intermediate of the Na,K-ATPase for cardiac steroid binding. Although the E2P has been thought to be the active form for binding, it cannot bind with the cardiac steroid in the presence of Na+ and in the absence of free Mg2+.

Two different phosphorylation-dephosphorylation cycles of Na,K-ATPase proteoliposomes accompanying Na+ transport in the absence of K+.

The phosphorylated intermediate (EP) of the Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme is composed of an ADP-sensitive K+-insensitive form (E1P), an ADP- and K+-sensitive form (E*P), and a K+-sensitive ADP-insensitive form (E2P). The composition of the intermediate varied with the cholesterol content of the lipid bilayer. The PL containing less than 30 mol % cholesterol (LCPL) formed E2P-rich EP in the presence of 10 mM Na+ on both sides at 15 degrees C, while the PL containing more than 35 mol % cholesterol (HCPL) formed E*P-rich EP under the same condition. In the presence of ionophore (monensin, nigericin, A23187), the HCPL formed E2P-rich EP as reported in the preceding paper. The turnover rate of Na-ATPase activity (the ratio of Na-ATPase to the EP level) in the LCPL was lower than that in the HCPL, and the addition of 20 microM monensin or A23187 to the HCPL reduced the Na-ATPase activity. The coupling ratio of Na+ influx (cellular efflux):Na+ efflux (cellular influx):ATP hydrolysis was 2.8:1.8:1 in the LCPL, although 1.6:0.6:1 in the HCPL. The coupling ratio of Na+ influx:ATP hydrolysis in the HCPL increased to 2.8:1 in the presence of A23187. Moreover, the increase of ATP concentration enhanced not only the Na-ATPase activity in the LCPL and HCPL with monensin but also the Na+ influx in the LCPL. This ATP enhancement was not found, however, in the HCPL without ionophores. The ADP enhancement of the Na+ influx was not observed in either the HCPL or the LCPL. We conclude from these observations that there are at least two different phosphorylation-dephosphorylation cycles (an E2P cycle and an E*P cycle) in the PL in the absence of K+. The E2P cycle transports three Na+ from the extravesicular (cytoplasmic) to the intravesicular (extracellular) side and two Na+ in the opposite direction per cycle and is similar to the ATP-dependent Na+-Na+ exchange system already reported (Blostein, R. (1983) J. Biol. Chem. 258, 7948-7953; Cornelius, F., and Skou, J. C. (1985) Biochim. Biophys. Acta 818, 211-221). However, the E*P cycle transports one Na+ from the extravesicular to the intravesicular side/cycle and has not yet been previously reported.

ADP- and K+-sensitive phosphorylated intermediate of Na,K-ATPase.

In the phosphoenzyme (EP) of the electric eel Na,K-ATPase, the sum of the ADP-sensitive EP and the K+-sensitive EP exceeds 150% of EP in the presence of 100 mM Na+. This unusual phenomenon can be explained by the formation of three phosphoenzymes: ADP-sensitive K+-insensitive (E1P), K+-sensitive ADP-insensitive (E2P), and ADP- and K+-sensitive (E*P) phosphoenzymes, as proposed by Nørby et al. (Nørby, J. G., Klodos, I., and Christiansen, N. O. (1983) J. Gen. Physiol. 82, 725-757). By applying a simple approximation method for the assay of E1P, E*P, and E2P, it was found that the phosphorylation of the enzyme was much faster than the conversion among each EP and the phosphoenzyme changed as E1NaATP----E1P----E*P----E2P. In the fragmental eel enzyme, the step of E*P to E2P was much slower than the step of E1P to E*P. In the steady state, the E1P was predominant above 400 mM Na+, whereas E*P and E2P were predominant between 60 and 300 mM Na+ and below 60 mM Na+, respectively. The characteristic difference of the eel enzyme from the beef brain enzyme and probably from the kidney enzyme seems to be that the dissociation constant of Na+ on the E1P-E*P equilibrium is higher than that on the E*P-E2P. The E*P and E1P both interacted with ADP to form ATP without formation of inorganic phosphate in the absence of free Mg2+. In the Na,K-ATPase proteoliposomes, the vesicle membrane interfered with the conversion of E1P to E2P, especially the change of E1P to E*P, and furthermore, the E1P content increased. This barrier effect was partially counteracted by monensin or carbonyl cyanide m-chlorophenylhydrazone. Oligomycin reacted with E1P and probably with E*P, therefore inhibiting their conversion to E2P and interaction with K+.

Quantitative evaluation of monocyte differentiation by electron microscopy: impairment of differentiation ability in monocytes from children with acute lymphoblastic leukaemia.

The differentiation of monocytes was evaluated quantitatively by electron microscopy and was analyzed in relation to the clinical features of childhood acute lymphoblastic leukaemia (ALL). The monocyte cellular size increased and cell organellae became mature after a 72-h culture. Phagolysosome formation developed markedly- and the nucleus became circular--accompanied by chromatin deconcentration. The differentiation degree of the cell organellae was indexed and classified as mature by electron microscopy. The indexes of nuclear shape, chromatin deconcentration, cytoplasmonuclear ratio and vacuole formation increased with time. The total index increased linearly with time dependence. These results indicate that the ultrastructural parameters and indexes allowed a quantitative assessment of cell organellae and cellular differentiation of the monocyte. At the acute phase of ALL, the degrees of cell organnellae and cellular differentiation were significantly lower than the control. The above findings suggest that monocytes from children with ALL have impaired differentiation ability, which results in defective function of macrophages.