Corn Oligopeptide Alleviates Nonalcoholic Fatty Liver Disease by Regulating the Sirtuin Signaling Pathway.

Nonalcoholic fatty liver disease (NAFLD) represents the most prevalent type of chronic liver disease, spanning from simple steatosis to nonalcoholic steatohepatitis (NASH). Corn oligopeptide (CP) is a functional peptide known for its diverse pharmacological effects on metabolism. In this study, we evaluated the protective activity of CP against fatty liver disease. Oral administration of CP significantly reduced body weight gain by 2.95%, serum cholesterol by 22.54%, and liver injury, as evidenced by a reduction of 32.19% in serum aspartate aminotransferase (AST) and 49.10% in alanine aminotransferase (ALT) levels in mice subjected to a high-fat diet (HFD). In a streptozotocin/HFD-induced NASH mouse model, CP attenuated body weight gain by 5.11%, liver injury (with a 34.15% decrease in AST and 11.43% decrease in ALT), and, to some extent, liver inflammation and fibrosis. Proteomic analysis revealed the modulation of oxidative phosphorylation and sirtuin (SIRT) signaling pathways by CP. Remarkably, CP selectively inhibited the hepatic expression of mitochondrial SIRT3 and SIRT5 in both HFD and NASH models. In summary, CP demonstrates a preventive effect against metabolic-stress-induced NAFLD progression by modulating oxidative stress and the SIRT signaling pathway, suggesting the potential of CP as a therapeutic agent for the treatment of NAFLD and advanced-stage NASH.

Clinical Activity and Exploratory Resistance Mechanism of Milademetan, an MDM2 Inhibitor, in Intimal Sarcoma with MDM2 Amplification: An Open-Label Phase Ib/II Study.

Intimal sarcoma is an extremely rare, life-threatening malignant neoplasm. Murine double minute 2 (MDM2) amplification is observed in >70% of intimal sarcomas. Milademetan, an MDM2 inhibitor, may provide clinical benefit in this patient population. We conducted a phase Ib/II study in patients with MDM2-amplified, wild-type TP53 intimal sarcoma as a substudy of a large nationwide registry for rare cancers in Japan. Milademetan (260 mg) was administered orally once daily for 3 days every 14 days, twice in a 28-day cycle. Of 11 patients enrolled, 10 were included in the efficacy analysis. Two patients (20%) showed durable responses for >15 months. Antitumor activity correlated with TWIST1 amplification (P = 0.028) and negatively with CDKN2A loss (P = 0.071). Acquired TP53 mutations were detected in sequential liquid biopsies as a novel exploratory resistance mechanism to milademetan. These results suggest that milademetan could be a potential therapeutic strategy for intimal sarcoma. SIGNIFICANCE: Strategies to optimize outcomes could include the use of new biomarkers (TWIST1 amplification and CDKN2A loss) to select patients with MDM2-amplified intimal sarcoma who might benefit from milademetan and combination with other targeted treatments. Sequential liquid biopsy of TP53 can be used to evaluate disease status during treatment with milademetan. See related commentary by Italiano, p. 1765. This article is highlighted in the In This Issue feature, p. 1749.

Evaluation of hepatic CYP3A enzyme activity using endogenous markers in lung cancer patients treated with cisplatin, dexamethasone, and aprepitant.

PURPOSE: Aprepitant is used with dexamethasone and 5-HT3 receptor antagonists as an antiemetic treatment for chemotherapy, including cisplatin. Aprepitant is a substrate of cytochrome P450 (CYP) 3A4 and is known to cause its inhibition and induction. In addition, dexamethasone is a CYP3A4 substrate that induces CYP3A4 and CYP3A5 expression. In this study, we aimed to quantitatively evaluate the profile of CYP3A activity using its endogenous markers in non-small cell lung cancer patients receiving a standard cisplatin regimen with antiemetics, including aprepitant. METHODS: Urinary 11ß-hydroxytestosterone (11ß-OHT)/testosterone concentration ratio and plasma 4ß-hydroxycholesterol (4ß-OHC) concentrations were measured before and after cisplatin treatment (days 1, 4, and 8). CYP3A5 was genotyped, and plasma aprepitant concentrations were measured on day 4 to examine its influence on CYP3A endogenous markers. RESULTS: The urinary 11ß-OHT/testosterone concentration ratio in the 35 patients included in this study increased by 2.65-fold and 1.21-fold on days 4 and 8 compared with day 1, respectively. Their plasma 4ß-OHC concentration increased by 1.46-fold and 1.66-fold, respectively. The mean plasma aprepitant concentration on day 4 was 1,451 ng/mL, which is far lower than its inhibitory constant. The allele frequencies of CYP3A5*1 and CYP3A5*3 were 0.229 and 0.771, respectively. In patients with the CYP3A5*1 allele, the plasma 4ß-OHC concentration was significantly lower at baseline but more potently increased with chemotherapy. CONCLUSION: CYP3A activity was significantly induced from day 4 to day 8 in patients receiving cisplatin and three antiemetic drugs.

Exacerbation of atrioventricular block associated with concomitant use of amlodipine and aprepitant in a lung cancer patient: A case report.

OBJECTIVE: To report a case of second-degree atrioventricular block associated with concomitant use of aprepitant and amlodipine. CASE: A 73-year-old man with lung cancer was treated with aprepitant for prophylactic use for the prevention of nausea and vomiting, concomitantly with cisplatin, gemcitabine, and an investigational drug (anti-epidermal growth factor receptor monoclonal antibody). He was diagnosed with first-degree atrioventricular block and was taking amlodipine for hypertension. During the first cycle of chemotherapy, 5 days after the start of aprepitant, he experienced Wenckebach second-degree atrioventricular block (Mobitz type I), and amlodipine was discontinued. After day 6, the atrioventricular block was not shown. According to the Naranjo adverse drug reaction scale, a score of 7 was obtained (causality: probable). In addition, using the Drug Interaction Probability Scale, a score of 6 was obtained (causality: probable). CONCLUSION: The drug-drug interaction between aprepitant and amlodipine was considered to have deteriorated his atrioventricular block, conceivably due to the inhibition of cytochrome P450 (CYP) 3A-mediated metabolism of amlodipine by aprepitant.

＜Editors' Choice＞ Efficacy of pectoral nerve block type-2 (Pecs II block) versus serratus plane block for postoperative analgesia in breast cancer surgery: a retrospective study.

Thoracic wall nerve blocks reduce postoperative acute pain after breast cancer surgery (BCS); however, their short-term effects and the most effective technique remain unclear. To compare the effects of pectoral nerve block type-2 (Pecs II block) and serratus plane block for postoperative short-term analgesia, we retrospectively reviewed 43 BCS patients who underwent Pecs II block (n=22) or serratus plane block (n=21). The primary outcome was the proportion of patients with no complaints of pain 2 months post-BCS. The odds ratio (OR) was assessed, adjusting for axillary lymph node dissection. The secondary outcomes were pain severity 24 hours and 2 months post-operation using the numerical rating scale score, and morphine consumption within 24 hours. The proportion of patients without pain 2 months post-BCS was significantly less with Pecs II block than in patients with serratus plane block (55% vs. 19%, adjusted OR, 5.04; 95% confidence interval, 1.26-20.07; P=0.02); the median [interquartile range] score for pain 2 months post-operation was also significantly lower with Pecs II block (Pecs II block 0.5 [0-1] vs. serratus plane block 1 [1-2]); P=0.03). Regarding post-BCS acute analgesia, the median [interquartile range] postoperative 24-hour pain score was 2 [1-3] and 3 [1.5-3.5], and the median morphine consumption within 24 hours was 1.5 [0.75-5.5] and 3 [1.5-10] mg in Pecs II block and serratus plane block (P=0.47 and P=0.11), respectively. This study suggests that Pecs II block prevents short-term post-BCS pain better than serratus plane block. However, further studies are needed in order to support this finding.

Individual optimal dose of amrubicin to prevent severe neutropenia in Japanese patients with lung cancer.

This study determined individual optimal amrubicin doses for Japanese patients with lung cancer after platinum-based treatment. We carried out population pharmacokinetic and pharmacodynamic modeling incorporating gene polymorphisms of metabolizing enzymes and transporters. Fifty patients with lung cancer, who were given 35-40 mg/m2 amrubicin on days 1-3 every 3-4 weeks, were enrolled. Mechanism-based modeling described relationships between the pharmacokinetics of amrubicin and absolute neutrophil counts. A population pharmacokinetic and pharmacodynamic model was developed for amrubicin and amrubicinol (active metabolite), connected by a delay compartment. The final model incorporated body surface area as a covariate of amrubicin and amrubicinol clearance and distribution volume. SLC28A3 single nucleotide polymorphism (rs7853758) was also incorporated as a constant covariate of the delay compartment of amrubicinol. Performance status was considered a covariate of pharmacokinetic (amrubicinol clearance) and pharmacodynamic (mean maturation time) parameters. Twenty-nine patients with grade 4 neutropenia showed higher amrubicinol area under the plasma concentration-time curve from 0 to 72 hours (AUC0-72 , P = .01) and shorter overall survival periods than other patients did (P = .01). Using the final population pharmacokinetic and pharmacodynamic model, median optimal dose to prevent grade 4 neutropenia aggravation was estimated at 22 (range, 8-40) mg/m2 for these 29 patients. We clarified correlations between area under the plasma concentration-time curve from 0 to 72 hours of amrubicinol and severity of neutropenia and survival of patients given amrubicin after platinum chemotherapy. This analysis revealed important amrubicin pharmacokinetic-pharmacodynamic covariates and provided useful information to predict patients who would require prophylactic granulocyte colony stimulating factor.

Excitatory Neuronal Hubs Configure Multisensory Integration of Slow Waves in Association Cortex.

Multisensory integration (MSI) is a fundamental emergent property of the mammalian brain. During MSI, perceptual information encoded in patterned activity is processed in multimodal association cortex. The systems-level neuronal dynamics that coordinate MSI, however, are unknown. Here, we demonstrate intrinsic hub-like network activity in the association cortex that regulates MSI. We engineered calcium reporter mouse lines based on the fluorescence resonance energy transfer sensor yellow cameleon (YC2.60) expressed in excitatory or inhibitory neurons. In medial and parietal association cortex, we observed spontaneous slow waves that self-organized into hubs defined by long-range excitatory and local inhibitory circuits. Unlike directional source/sink-like flows in sensory areas, medial/parietal excitatory and inhibitory hubs had net-zero balanced inputs. Remarkably, multisensory stimulation triggered rapid phase-locking mainly of excitatory hub activity persisting for seconds after the stimulus offset. Therefore, association cortex tends to form balanced excitatory networks that configure slow-wave phase-locking for MSI. VIDEO ABSTRACT.

Netrin-G1 regulates fear-like and anxiety-like behaviors in dissociable neural circuits.

In vertebrate mammals, distributed neural circuits in the brain are involved in emotion-related behavior. Netrin-G1 is a glycosyl-phosphatidylinositol-anchored synaptic adhesion molecule whose deficiency results in impaired fear-like and anxiety-like behaviors under specific circumstances. To understand the cell type and circuit specificity of these responses, we generated netrin-G1 conditional knockout mice with loss of expression in cortical excitatory neurons, inhibitory neurons, or thalamic neurons. Genetic deletion of netrin-G1 in cortical excitatory neurons resulted in altered anxiety-like behavior, but intact fear-like behavior, whereas loss of netrin-G1 in inhibitory neurons resulted in attenuated fear-like behavior, but intact anxiety-like behavior. These data indicate a remarkable double dissociation of fear-like and anxiety-like behaviors involving netrin-G1 in excitatory and inhibitory neurons, respectively. Our findings support a crucial role for netrin-G1 in dissociable neural circuits for the modulation of emotion-related behaviors, and provide genetic models for investigating the mechanisms underlying the dissociation. The results also suggest the involvement of glycosyl-phosphatidylinositol-anchored synaptic adhesion molecules in the development and pathogenesis of emotion-related behavior.

Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies.

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.

Cells of a common developmental origin regulate REM/non-REM sleep and wakefulness in mice.

Mammalian sleep comprises rapid eye movement (REM) sleep and non-REM (NREM) sleep. To functionally isolate from the complex mixture of neurons populating the brainstem pons those involved in switching between REM and NREM sleep, we chemogenetically manipulated neurons of a specific embryonic cell lineage in mice. We identified excitatory glutamatergic neurons that inhibit REM sleep and promote NREM sleep. These neurons shared a common developmental origin with neurons promoting wakefulness; both derived from a pool of proneural hindbrain cells expressing Atoh1 at embryonic day 10.5. We also identified inhibitory γ-aminobutyric acid-releasing neurons that act downstream to inhibit REM sleep. Artificial reduction or prolongation of REM sleep in turn affected slow-wave activity during subsequent NREM sleep, implicating REM sleep in the regulation of NREM sleep.

Astrocytic Ca2+ signals are required for the functional integrity of tripartite synapses.

BACKGROUND: Neuronal activity alters calcium ion (Ca2+) dynamics in astrocytes, but the physiologic relevance of these changes is controversial. To examine this issue further, we generated an inducible transgenic mouse model in which the expression of an inositol 1,4,5-trisphosphate absorbent, "IP3 sponge", attenuates astrocytic Ca2+ signaling. RESULTS: Attenuated Ca2+ activity correlated with reduced astrocytic coverage of asymmetric synapses in the hippocampal CA1 region in these animals. The decreased astrocytic 'protection' of the synapses facilitated glutamate 'spillover', which was reflected by prolonged glutamate transporter currents in stratum radiatum astrocytes and enhanced N-methyl-D-aspartate receptor currents in CA1 pyramidal neurons in response to burst stimulation. These mice also exhibited behavioral impairments in spatial reference memory and remote contextual fear memory, in which hippocampal circuits are involved. CONCLUSIONS: Our findings suggest that IP3-mediated astrocytic Ca2+ signaling correlates with the formation of functional tripartite synapses in the hippocampus.

Pharmacokinetic and pharmacodynamic study on amrubicin and amrubicinol in Japanese patients with lung cancer.

PURPOSE: The pharmacokinetic (PK)-pharmacodynamic (PD) relationship of amrubicin and its active metabolite, amrubicinol, has only been evaluated using trough levels of these agents since the full PK profiles not yet been clarified so far. This study was performed to analyze the full PK profiles of amrubicin and amrubicinol and to evaluate their toxicity-PK relationships in Japanese patients. METHODS: Amrubicin (35-40 mg/m(2)) was administered to 21 lung cancer patients on days 1-3 every 3-4 weeks. Fourteen blood samples were obtained per patient over the course of 3 administration days. The plasma concentrations of amrubicin and amrubicinol were quantitated by HPLC, and the relationships between PK parameters of these compounds and hematological toxicities were evaluated. RESULTS: The overall PK profiles of amrubicin and amrubicinol were well characterized using a 3-compartment model and a 1-compartment model with a first-order metabolic process, respectively. The major toxicities were hematological. The clearance of amrubicinol was significantly correlated with grade 4 neutropenia (P = 0.01). The percentage decreases in the neutrophil count, hemoglobin level and platelet count were well correlated with the amrubicinol AUC. CONCLUSION: The pharmacokinetic profiles of amrubicin and amrubicinol were clarified, and the subsequent PK-PD analyses indicate that the clearance of amrubicinol is the major determinant of neutropenia.

Strong antibody reaction against glycosphingolipids injected in liposome-embedded forms in beta3GN-T5 knockout mice.

It is known that mutant mice of the beta-1,3-N-acetylglucosaminyltransferase gene (beta3Gn-T5) respond well to T-cell dependent and independent antigens. Here, we examined the effectiveness of anti-ganglioside antibody generation by immunization of beta3Gn-T5 mutant mice with liposome-embedded glycosphingolipids such as GD1a and GT1b. Consequently, the mutant mice showed a more efficient generation of anti-GD1a or anti-GT1b antibodies than wild-type mice in an enzyme-linked immunosorbent assay using sera during immunization. Thus, the beta3Gn-T5 deficient mutant mice proved more responsive than wild-type mice to not only protein antigens, but also to carbohydrates in glycolipids. Furthermore, about 50% of monoclonal antibodies generated using splenocytes of the immunized mutant mice were of the IgG class. Besides general high responsiveness to proteins and glycolipids, it could be expected that the mutant mice of beta3Gn-T5 would be useful in the generation of monoclonal antibodies towards lacto-/neolacto-series glycolipids, since these mutants lack lacto-/neolacto-series glycolipids. In fact, they showed a good serum response in immuno-fluorescence assay with cultured living cells when immunized by glycolipids extracted from ovarian cancer cell lines. These results suggested that beta3Gn-T5 mutant mice are useful for the generation of anti-glycolipid antigens with lacto-/neolacto-core structures expressed in cancer cells.

Efficient generation of useful monoclonal antibodies reactive with globotriaosylceramide using knockout mice lacking Gb3/CD77 synthase.

Efficient generation of useful monoclonal antibodies (mAbs) with high performance in cancer therapeutics has been expected. Generation of mAbs reactive with globotriaosylceramide (Gb3/CD77) was compared between A/J mice and Gb3/CD77 synthase-deficient (A4GalT-knockout) mice by immunizing Gb3-liposome. Specificity and functions of established antibodies were examined by ELISA, TLC- immunostaining, cytotoxicity of cancer cells and immunoblotting. Compared with results with conventional mice, better generation of mAbs with higher functions has been achieved with A4GalT-knockout mice, i.e. acquisition of IgG class antibodies, activities in antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and aggregation activity toward a Burkitt's lymphoma line Ramos. Binding of mAb k52 induced tyrosine phosphorylation of several proteins in Ramos cells. One of the strongest phosphorylation bands turned out to be c-Cbl. Pretreatment of B cell lines with mAbs resulted in the attenuation of BCR-mimicking signaling. All these results suggested that A4GalT-knockout mice are very useful to generate mAbs against globo-series glycolipids, and that suppressive signaling pathway driven by endogenous Gb3-ligand molecules might be present in B cells.

Simple and sensitive HPLC method for determination of amrubicin and amrubicinol in human plasma: application to a clinical pharmacokinetic study.

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for determination of amrubicin and its metabolite amrubicinol in human plasma. After protein precipitation with methanol without evaporation procedure, large volume samples were injected and separated by two monolithic columns with a guard column. The mobile phase consisted of tetrahydrofuran-dioxane-water (containing 2.3 mM acetic acid and 4 mM sodium 1-octanesulfonate; 2:6:15, v/v/v). Wavelengths of fluorescence detection were set at 480 nm for excitation and 550 nm for detection. Under these conditions, linearity was confirmed in the 2.5-5000 ng/mL concentration range of both compounds. The intra- and inter-day precision and intra- and inter-day accuracy for both compounds were less than 10%. The method was successfully applied to a clinical pharmacokinetic study of amrubicin and amrubicinol in cancer patients.

Association between tinnitus retraining therapy and a tinnitus control instrument.

OBJECTIVE: Tinnitus retraining therapy (TRT), which is an adaptation therapy for tinnitus based on the neurophysiological model proposed by Jastreboff in 1990,consists of directive counseling and acoustic therapy with a tinnitus control instrument (TCI) or other devices. For the past 5 years, our hospital has administered TRT characterized by the use of a TCI. METHOD: In this study, we reviewed the clinical course of patients with tinnitus who presented to our outpatient clinic for tinnitus and hearing loss during the 3-year period from April 2004 to March 2007 and underwent TRT with a TCI. Among 188 patients with tinnitus (105 males and 83 females), 88 patients (51 males and 37 females, excluding dropouts) who purchased a TCI and continued therapy were included in the study. RESULTS: Significant improvement in Tinnitus Handicap Inventory (THI) and Visual Analogue Scale (VAS) scores was found as early as 1 month of treatment and later compared with those on initial examination, suggesting that TRT with a TCI may be an effective treatment for tinnitus. Among the noises generated by the TCI, the sound pressure output from the TCI was set at just below tinnitus loudness level both of the first adjustment and the second adjustment. Speech noise and white noise were frequently selected, whereas high-frequency noise and pink noise were infrequently selected. Speech noise was most frequently selected at the first adjustment, and the number of patients selecting white noise increased at the second adjustment. The results that we compared the two also revealed that the mean hearing level and tinnitus loudness levels were higher in the white noise group than in the speech noise group, which suggested that the inner ear disorder was more harder in the white noise group. Both the THI score and VAS grade improved after 1 month of treatment in the speech noise group, whereas improvement in these parameters was observed in the white noise group after 6 months of treatment. These results suggest that it took much longer the patients in the white noise group to improve. CONCLUSION: : Significant improvement in THI and VAS scores was found as early as 1 month of treatment and later compared with those on initial examination, suggesting that TRT with a TCI may be an effective treatment for tinnitus. It resulted that many patients chose the speech noise or the white noise. And also it was indicated that noise generators set at just below mixing point with tinnitus are more effective. In this study, however, speech noise was often selected probably because of the reduced output at high frequencies and the level of comfort. As white noise produces greater sound volume, patients tended to switch from other therapeutic sound to white noise at the second adjustment. These findings may help administer acoustic therapy in the future.

Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling.

The ephrin/Eph system plays a central role in neuronal circuit formation; however, its downstream effectors are poorly understood. Here we show that alpha-chimerin Rac GTPase-activating protein mediates ephrinB3/EphA4 forward signaling. We discovered a spontaneous mouse mutation, miffy (mfy), which results in a rabbit-like hopping gait, impaired corticospinal axon guidance, and abnormal spinal central pattern generators. Using positional cloning, transgene rescue, and gene targeting, we demonstrated that loss of alpha-chimerin leads to mfy phenotypes similar to those of EphA4(-/-) and ephrinB3(-/-) mice. alpha-chimerin interacts with EphA4 and, in response to ephrinB3/EphA4 signaling, inactivates Rac, which is a positive regulator of process outgrowth. Moreover, downregulation of alpha-chimerin suppresses ephrinB3-induced growth cone collapse in cultured neurons. Our findings indicate that ephrinB3/EphA4 signaling prevents growth cone extension in motor circuit formation via alpha-chimerin-induced inactivation of Rac. They also highlight the role of a Rho family GTPase-activating protein as a key mediator of ephrin/Eph signaling.

Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice.

The ability to restrict gene expression or disruption to specific regions of the brain would enhance understanding of the molecular basis for brain development and function. For this purpose, brain region-restricted promoters are essential. Here we report the isolation of a DNA fragment containing the Emx1 gene promoter, which is responsible for dorsal telencephalon-specific expression. The Cre recombinase gene was inserted into a mouse PAC (P1-derived artificial chromosome) Emx1-locus clone (PAC-Emx1#1 clone) and utilized to generate three transgenic mouse lines. In all three lines, especially Tg3, Cre-mediated recombination was highly restricted to Emx1-expressing cell lineages, from embryonic stages to adulthood. Immunohistochemical analyses showed that Cre protein is expressed in the dorsal telencephalon in all three lines in adulthood. Thus, the PAC-Emx1#1 clone contains essentially all regulatory elements necessary for Emx1 gene expression. Our results suggest that Emx1-Cre Tg3 mice and the PAC-Emx1#1 clone constitute powerful tools for dorsal telencephalon-specific gene manipulation.