Crucial role of TFAP2B in the nervous system for regulating NREM sleep.

The AP-2 transcription factors are crucial for regulating sleep in both vertebrate and invertebrate animals. In mice, loss of function of the transcription factor AP-2ß (TFAP2B) reduces non-rapid eye movement (NREM) sleep. When and where TFAP2B functions, however, is unclear. Here, we used the Cre-loxP system to generate mice in which Tfap2b was specifically deleted in the nervous system during development and mice in which neuronal Tfap2b was specifically deleted postnatally. Both types of mice exhibited reduced NREM sleep, but the nervous system-specific deletion of Tfap2b resulted in more severe sleep phenotypes accompanied by defective light entrainment of the circadian clock and stereotypic jumping behavior. These findings indicate that TFAP2B in postnatal neurons functions at least partly in sleep regulation and imply that TFAP2B also functions either at earlier stages or in additional cell types within the nervous system.

"Fruiting Liquid" of Mushroom-Forming Fungi, A Novel Source of Bioactive Compounds - Fruiting-Body Inducer and HIF and Axl Inhibitors.

In general, mushroom-forming fungi secrete liquid on the surface of mycelia just before fruiting-body formation. However, no researchers in mushroom science have paid attention to the liquid until now. We formulated a hypothesis that the liquid plays an important role(s) in the formation of the fruiting body and produces various bioactive compounds and named it the "fruiting liquid (FL)". Four novel compounds (1-4) were isolated from FL of Hypholoma lateritium and Hericium erinaceus. The structures of 1-4 except for their stereochemistry were determined by interpretation of MS and NMR data. The absolute configurations of compounds 1-4 were determined by quantum chemical calculation of the ECD spectrum, by single-crystal X-ray diffraction analyses, or by chemical syntheses. Compounds 1, 3, and 4 induced fruiting body formation of Flammulina velutipes. Compound 4 inhibited the activity of hypoxia-inducible factor, and compounds 2-4 suppressed receptor tyrosine kinase (Axl) expression.

Pemafibrate prevents choroidal neovascularization in a mouse model of neovascular age-related macular degeneration.

Background: Pathological choroidal neovascularization (CNV) is one of the major causes of visual impairment in neovascular age-related macular degeneration (AMD). CNV has been suppressed by using anti-vascular endothelial growth factor (VEGF) antibodies. However, some clinical cases have demonstrated the failure of anti-VEGF therapies. Furthermore, anti-VEGF agents might induce the development of ocular atrophy. Recently, peroxisome proliferator-activated receptor alpha (PPARα) activation using pemafibrate treatment was suggested as one of the promising therapeutic targets in the prevention of ocular ischemia. However, the preventive role of pemafibrate remains unclear in CNV. We aimed to examine the preventive role of pemafibrate on laser-induced pathological CNV. Methods: Adult male C57BL/6 mice were orally supplied pemafibrate (0.5 mg/kg) for four days, followed by laser irradiation. Then, pemafibrate was consecutively given to mice with the same condition. CNV was visualized with isolectin-IB4. The eye (retina and/or retinal pigment epithelium [RPE]-choroid), liver, and serum were used for biomolecular analyses. Results: We found that pemafibrate administration suppressed CNV volumes. Pemafibrate administration activated PPARα downstream genes in the liver and eye (especially, RPE-choroid). Furthermore, pemafibrate administration elevated serum fibroblast growth factor 21 levels and reduced serum levels of triglycerides. Conclusions: Our data suggest a promising pemafibrate therapy for suppressing CNV in AMD.

Fully automatic REM sleep stage-specific intervention systems using single EEG in mice.

Sleep stage-specific intervention is widely used to elucidate the functions of sleep and their underlying mechanisms. For this intervention, it is imperative to accurately classify rapid-eye-movement (REM) sleep. However, the proof of fully automatic real-time REM sleep classification in vivo has not been obtained in mice. Here, we report the in vivo implementation of a system that classifies sleep stages in real-time from a single-channel electroencephalogram (EEG). It enabled REM sleep-specific intervention with 90 % sensitivity and 86 % precision without prior configuration to each mouse. We further derived systems capable of classification with higher frequency sampling and time resolution. This attach-and-go sleep staging system provides a fully automatic accurate and scalable tool for investigating the functions of sleep.

Modulation of Hypoxia-Inducible Factors and Vascular Endothelial Growth Factor Expressions by Superfood Camu-Camu (Myrciaria dubia) Treatment in ARPE-19 and Fetal Human RPE Cells.

Background: Anti-vascular endothelial growth factor (anti-VEGF) therapy via intravitreal injection is an effective treatment for patients with abnormal ocular neovascularization, such as age-related macular degeneration (AMD) and diabetic macular edema (DME). However, prolonged and frequent anti-VEGF treatment is associated with a risk of local and systemic adverse events, including geographic atrophy, cerebrovascular disease, and death. Furthermore, some patients do not adequately respond to anti-VEGF therapy. Hypoxia-inducible factor (HIF) is a transcription factor that controls the expression of hypoxia-responsive genes involved in angiogenesis, inflammation, and metabolism. The HIF/VEGF pathway plays an important role in neovascularization, and the inhibition of HIF activation could be an effective biomolecular target for neovascular diseases. The demand for disease prevention or treatment using functional foods such as superfoods has increased in recent years. Few reports to date have focused on the antineovascular effects of superfoods in the retinal pigment epithelium (RPE). In light of the growing demand for functional foods, we aimed to find novel HIF inhibitors from superfoods worked in RPE cells, which could be an adjuvant for anti-VEGF therapy. Methods: Seven superfoods were examined to identify novel HIF inhibitor candidates using luciferase assay screening. We used the human RPE cell line ARPE-19 and fetal human RPE (fhRPE) to investigate the biomolecular actions of novel HIF inhibitors using quantitative PCR and western blotting. Results: Under CoCl2-induced pseudohypoxic condition and 1% oxygen hypoxic incubation, camu-camu (Myrciaria dubia) showed HIF inhibitory effects determined by luciferase assays. Camu-camu downregulated HIF-1α and VEGFA mRNA expressions in a concentration-dependent manner. Camu-camu also inhibited HIF-1α protein expressions, and its inhibitory effect was greater than that of vitamin C, which is present at high levels in camu-camu. Conclusion: The camu-camu extract suppressed the activation of HIF and VEGF in RPE cells. This could assist anti-VEGF therapy in patients with abnormal ocular neovascularization.

Axl, Immune Checkpoint Molecules and HIF Inhibitors from the Culture Broth of Lepista luscina.

Two compounds 1 and 2 were isolated from the culture broth of Lepista luscina. This is the first time that compound 1 was isolated from a natural source. The structure of compound 1 was identified via 1D and 2D NMR and HRESIMS data. Compounds 1 and 2 along with 8-nitrotryptanthrin (4) were evaluated for their biological activities using the A549 lung cancer cell line. As a result, 1 and 2 inhibited the expression of Axl and immune checkpoint molecules. In addition, compounds 1, 2 and 4 were tested for HIF inhibitory activity. Compound 2 demonstrated statistically significant HIF inhibitory effects on NIH3T3 cells and 1 and 2 against ARPE19 cells.

Retinal degeneration induced in a mouse model of ischemia-reperfusion injury and its management by pemafibrate treatment.

Retinal ischemia-reperfusion (I/R) injury is a common cause of visual impairment. To date, no effective treatment is available for retinal I/R injury. In addition, the precise pathological mechanisms still need to be established. Recently, pemafibrate, a peroxisome proliferator-activated receptor α (PPARα) modulator, was shown to be a promising drug for retinal ischemia. However, the role of pemafibrate in preventing retinal I/R injury has not been documented. Here, we investigated how retinal degeneration occurs in a mouse model of retinal I/R injury by elevation of intraocular pressure and examined whether pemafibrate could be beneficial against retinal degeneration. Adult mice were orally administered pemafibrate (0.5 mg/kg/day) for 4 days, followed by retinal I/R injury. The mice were continuously administered pemafibrate once every day until the end of the experiments. Retinal functional changes were measured using electroretinography. Retina, liver, and serum samples were used for western blotting, quantitative PCR, immunohistochemistry, or enzyme linked immunosorbent assay. Retinal degeneration induced by retinal inflammation was prevented by pemafibrate administration. Pemafibrate administration increased the hepatic PPARα target gene expression and serum levels of fibroblast growth factor 21, a neuroprotective molecule in the eye. The expression of hypoxia-response and pro-and anti-apoptotic/inflammatory genes increased in the retina following retinal I/R injury; however, these changes were modulated by pemafibrate administration. In conclusion, pemafibrate is a promising preventive drug for ischemic retinopathies.

Vascular endothelial growth factor from retinal pigment epithelium is essential in choriocapillaris and axial length maintenance.

Myopia, which prevalence is rapidly increasing, causes visual impairment; however, the onset mechanism of pathological axial length (AL) elongation remains unclear. A highly vascularized choroid between the retinal pigment epithelium (RPE) and sclera not only maintains physiological activities, but also contributes to ocular development and growth regulation. Vascular endothelial growth factor (VEGF) secreted from the RPE to the choroid is essential for retinal function and maintenance of the choriocapillaris. Herein, we demonstrated that the loss of VEGF secreted from the RPE caused abnormal choriocapillaris development and AL elongation, with features similar to those of the lens-induced myopia (LIM) mouse model, whereas VEGF overexpression by knocking-out von Hippel-Lindau (VHL) specific to the RPE expands the choriocapillaris and shortens the AL. Additionally, LDL Receptor Related Protein 2 (LRP2) deletion in the RPE downregulated VEGF expression and leads to pathological AL elongation. Furthermore, high-myopia patients without choriocapillaris demonstrated longer ALs than did those with preserved choriocapillaris. These results suggest that physiological secretion of VEGF from the RPE is required for proper AL development by maintaining the choriocapillaris. The pinpoint application of VEGF to the choriocapillaris may become a potential intervention for the prevention and treatment of axial myopia progression.

HIF Inhibition Therapy in Ocular Diseases.

The uncontrolled growth of blood vessels is a major pathological factor in human eye diseases that can result in blindness. This effect is termed ocular neovascularization and is seen in diabetic retinopathy, age-related macular degeneration, glaucoma and retinopathy of prematurity. Current treatments for these diseases include laser photocoagulation, topical injection of corticosteroids, intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) agents and vitreoretinal surgery. Although strategies to inhibit VEGF have proved to be dramatically successful in some clinical studies, there remains the possibility of significant adverse effects regarding the blockade of crucial physiological roles of VEGF and the invasive nature of the treatments. Moreover, it is evident that other pro-angiogenic factors also play important roles in the development of these diseases, as seen in cases in which anti-VEGF therapies have failed. Therefore, new types of effective treatments are required. In this review, we discuss a promising strategy for the treatment of ocular neovascular diseases, i.e., the inhibition of hypoxia-inducible factor (HIF), a master regulator of angiogenesis. We also summarize promising recently investigated HIF inhibitors as treatments for ocular diseases. This review will facilitate more comprehensive approaches to understanding the protective aspects of HIF inhibition in the prevention of ocular diseases.

Impaired wakefulness and rapid eye movement sleep in dopamine-deficient mice.

Despite the established roles of the dopaminergic system in promoting arousal, the effects of loss of dopamine on the patterns of sleep and wakefulness remain elusive. Here, we examined the sleep architecture of dopamine-deficient (DD) mice, which were previously developed by global knockout of tyrosine hydroxylase and its specific rescue in noradrenergic and adrenergic neurons. We found that DD mice have reduced time spent in wakefulness. Unexpectedly, DD mice also exhibited a marked reduction in the time spent in rapid eye movement (REM) sleep. The electroencephalogram power spectrum of all vigilance states in DD mice were also affected. These results support the current understanding of the critical roles of the dopaminergic system in maintaining wakefulness and also implicate its previously unknown effects on REM sleep.

Retinal Degeneration in a Murine Model of Retinal Ischemia by Unilateral Common Carotid Artery Occlusion.

Retinal degeneration is a progressive retinal damage in ocular vascular diseases. There are several reasons for this, such as occlusion of arteries or veins, diabetic retinopathy, or hereditary retinal diseases. To study pathological mechanisms of retinal degeneration, it is required to develop experimentally reproducible and clinically relevant models. In our previous studies, we developed a murine model of retinal hypoperfusion by unilateral common carotid artery occlusion (UCCAO) which mimics the pathophysiology of ocular ischemic syndrome (OIS) in humans, and described broad pathological mechanisms in the retina after UCCAO. However, there still remain missing pieces of the ocular pathologic process by UCCAO. In this study, we examined those unfound mechanisms. UCCAO was performed on adult mice. Ocular dysfunctions, histological deficits, and inflammation were examined after UCCAO, compared with sham-operated mice. Evaluation values were analyzed by electrophysiological, histological, and molecular biological methods. Eyelid drooping was permanently seen after UCCAO. Induction time point of acute reversible cataract under anesthesia was shortened. Retinal/visual dysfunctions were detected 2-4 weeks after UCCAO. Specifically, scotopic b-wave was more affected than a-wave, with the dysfunction of photopic b-wave. Impaired oscillatory potentials and visual evoked potential were constantly observed. Pathological Müller gliosis/inflammation was featured with NeuN-positive cell loss in the ganglion cell layer. Axial length, intraocular pressure, pupillary light reflex, and retinal pigment epithelium/choroidal thickness were not changed by UCCAO. A murine model of retinal ischemia by UCCAO can be useful for studying a series of degenerative process in the ischemic retina.

Sleep Architecture in Mice Is Shaped by the Transcription Factor AP-2ß.

The molecular mechanism regulating sleep largely remains to be elucidated. In humans, families that carry mutations in TFAP2B, which encodes the transcription factor AP-2ß, self-reported sleep abnormalities such as short-sleep and parasomnia. Notably, AP-2 transcription factors play essential roles in sleep regulation in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster Thus, AP-2 transcription factors might have a conserved role in sleep regulation across the animal phyla. However, direct evidence supporting the involvement of TFAP2B in mammalian sleep was lacking. In this study, by using the CRISPR/Cas9 technology, we generated two Tfap2b mutant mouse strains, Tfap2bK144 and Tfap2bK145 , each harboring a single-nucleotide mutation within the introns of Tfap2b mimicking the mutations in two human kindreds that self-reported sleep abnormalities. The effects of these mutations were compared with those of a Tfap2b knockout allele (Tfap2b-). The protein expression level of TFAP2B in the embryonic brain was reduced to about half in Tfap2b+/- mice and was further reduced in Tfap2b-/- mice. By contrast, the protein expression level was normal in Tfap2bK145/+ mice but was reduced in Tfap2bK145/K145 mice to a similar extent as Tfap2b-/- mice. Tfap2bK144/+ and Tfap2bK144/K144 showed normal protein expression levels. Tfap2b+/- female mice showed increased wakefulness time and decreased nonrapid eye movement sleep (NREMS) time. By contrast, Tfap2bK145/+ female mice showed an apparently normal amount of sleep but instead exhibited fragmented NREMS, whereas Tfap2bK144/+ male mice showed reduced NREMS time specifically in the dark phase. Finally, in the adult brain, Tfap2b-LacZ expression was detected in the superior colliculus, locus coeruleus, cerebellum, and the nucleus of solitary tract. These findings provide direct evidence that TFAP2B influences NREMS amounts in mice and also show that different mutations in Tfap2b can lead to diverse effects on sleep architecture.

Multiple Metastasizing Pleomorphic Adenomas of the Lung.

A 40-year-old woman was referred to our hospital with abnormal findings on chest X-ray. Her medical history was remarkable in that she had presented with a pleomorphic adenoma in the right parotid gland treated by surgical removal approximately 12 years previously. Chest computed tomography showed well-defined non-calcified nodules of the bilateral lobes, so she underwent segmentectomy of the right upper lobe and middle lobe. The histopathological diagnosis was metastasizing pleomorphic adenoma of the lung, a rare entity.

Laser desorption ionization of stilbenes in crystalline sponge.

The laser desorption (LD) ionization of three stilbenes in the nano porous metal-organic frameworks called "crystalline sponge" is demonstrated. The analyte position in the pore and the interaction between the analyte and the framework that functions as a matrix are discussed based on the results of single- crystal X-ray analysis. It is confirmed that the analyte/ligand ratio of 1:2 is sufficient for the analyte ionization. This method makes it possible to visualize hot spots on a target plate to be irradiated. That the sample requirement is dramatically reduced to the order of femtomoles is also an advantage. The relationship between laser interaction, analyte position in the pore, and analyte/ligand ratio is discussed as a new ionization field to elucidate the molecular structure of the analyte by LD ionization mass spectrometry.

Comprehensive extraction study using N,N-dioctyldiglycolamic acid.

We report on the acid dissociation constants (Ka) of diglycolamic acid-type ligands together with comprehensive data on the extraction performance of N,N-dioctyldiglycolamic acid (DODGAA) for 54 metal ions. The pKa of the diglycolamic acid framework was determined to be 3.54 ± 0.03 in water (0.1 M LiCl, 25°C) by potentiometric titration, indicating that DODGAA is strongly acidic compared with acetic acid. DODGAA can quantitatively transfer various metal ions among the 54 metal ions through a proton-exchange reaction, and provides excellent extraction performance and separation ability for rare-earth metal ions, In(III), Fe(III), Hg(II), and Pb(II) among the 54 metal ions.

Extraction behavior and selective separation of lead(II) using N,N-dioctyldiglycol amic acid.

Selective separation of lead ions (Pb(2+)) from aqueous solutions containing multiple divalent metal ions (Pb(2+), Cu(2+), Cd(2+), Zn(2+), Mn(2+), Co(2+), and Ni(2+)) was investigated using liquid-liquid extraction. N,N-Dioctyldiglycol amic acid (DODGAA) enabled quantitative extraction and efficient separation of Pb(2+) from the metal ion mixture under mildly acidic conditions. Compared with conventional commercial extractants, DODGAA provided better extraction and excellent selectivity for Pb(2+). The extraction of Pb(2+) with DODGAA proceeded through a proton-exchange reaction and formed a 1:2 complex, Pb(DODGAA)(2). The Pb(2+) was readily stripped from the extracting phase under acidic conditions, and the organic solution with DODGAA could be recycled.

Structural study on 2,2'-(methylimino)bis(N,N-dioctylacetamide) complex with Re(VII)O4- and Tc(VII)O4- by 1H NMR, EXAFS, and IR spectroscopy.

The structures of the complex of 2,2'-(methylimino)bis(N,N-dioctylacetamide) (MIDOA) with M(VII)O(4)(-) (M = Re and Tc), which were prepared by liquid-liquid solvent extraction, were investigated by using (1)H nuclear magnetic resonance (NMR), extended X-ray absorption fine structure (EXAFS), and infrared (IR) spectroscopy. The (1)H NMR spectra of the complex of MIDOA with Re(VII)O(4)(-) prepared in the organic solution suggest the transfer of a proton from aqueous to organic solution and the formation of the H(+)MIDOA ion. The EXAFS spectra of the complexes of H(+)MIDOA with Re(VII)O(4)(-) and Tc(VII)O(4)(-) show only the M-O coordination of the aquo complexes, suggesting that the chemical state of M(VII)O(4)(-) was unchanged during the extraction process. The results from (1)H NMR and EXAFS, therefore, provide evidence of M(VII)O(4)(-)···H(+)MIDOA complex formation in the organic solution. The IR spectra of Re(VII)O(4)(-)···H(+)MIDOA and Tc(VII)O(4)(-)···H(+)MIDOA were analyzed based on the structures and the IR spectra that were calculated at the B3LYP/cc-pVDZ level. Comparison of the observed and calculated IR spectra demonstrates that an intramolecular hydrogen bond is formed in H(+)MIDOA, and the M(VII)O(4)(-) ion interacts with H(+)MIDOA through multiple C-H(n)···O hydrogen bonds.