Visualization of mitochondria with green fluorescent protein in cultured fibroblasts from patients with mitochondrial diseases

cDNAs for green fluorescent protein (GFP) and for a GFP fusion protein containing the presequence of human ornithine transcarbamylase (pOTC-GFP) were transfected into cultured human fibroblasts. GFP cDNA gave diffuse fluorescence throughout the cytoplasm and the nucleus, whereas pOTC-GFP cDNA gave mitochondria-associated fluorescence. Fluorescent mitochondrial structures could be classified into five patterns: thread-like mitochondria, fine thread-like ones, rod-like ones, granular ones, and granular ones with weak cytosolic fluorescence. pOTC-GFP mutants resulted in a loss of mitochondrial fluorescence and an appearance of weak fluorescence throughout the cytoplasm. pOTC-GFP cDNA was transfected into fibroblasts from patients with various mitochondrial diseases. Higher ratios of fibroblasts with granular mitochondria and those with fine thread-like ones were observed in a patient with Reye's syndrome and a patient with Kearns-Sayre syndrome. Weak cytosolic fluorescence was sometimes observed in fibroblasts from these patients. This method will be useful to analyze mitochondrial structural alterations and disorders of mitochondrial protein import.     

Effects of short- and long-term dexamethasone treatment on growth and growth hormone (GH)-releasing hormone (GRH)-GH-insulin-like growth factor-I axis in conscious rats

Although the inhibitory effects of a chronic excess of glucocorticoids (GC) on body growth and GH secretion are well established, the mechanisms involved remain unclear. In this study, we examined the chronic effects of a high dose of dexamethasone (DEX) on spontaneous GH secretion and insulin-like growth factor (IGF)-I in conscious rats. The animals were given daily i.p. injections of DEX (200 microg/day) for either one or four weeks. Body growth assessed by tibia length and serum IGF-I levels was significantly inhibited 1 week after treatment. By contrast, spontaneous GH secretion was not altered 1 week after the treatment. Neither hypothalamic GRH and somtatostain mRNA levels nor GH responses to GRH from single somatotropes were affected 1 week after the treatment. Four weeks after DEX treatment, body growth of the rats was noticeably suppressed. Interestingly, spontaneous GH secretion, hypothalamic GRH mRNA levels and GH responses to GRH were all inhibited 4 weeks after treatment. Pituitary GRH receptor mRNA levels were not altered 1 week after treatment, but increased after 4 weeks. These results indicate that a high dose of DEX initially impairs IGF-I production and subsequently inhibits spontaneous GH secretion in rats. Inhibition of spontaneous GH secretion resulting from chronic GC excess is due, at least in part, to the impairment of hypothalamic GRH synthesis and pituitary GH responsiveness. An increase in the pituitary GRH receptor may be caused by decreased GRH secretion.     

Screening of Inhibitors Targeting Heat Shock Protein 47 Involved in the Development of Idiopathic Pulmonary Fibrosis

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is causally related to fibrotic diseases, including idiopathic pulmonary fibrosis. The identification of Compounds that interfere with the HSP47-collagen interaction is essential for the development of relevant therapeutics. Herein, we prepared human HSP47 as a soluble fusion protein expressed in E. coli and established an assay system for HSP47 inhibitor screening. We screened a natural and synthetic Compound library established at Nagasaki University. Among 1023 Compounds, 13 exhibited inhibitory activity against human HSP47, of which three inhibited its function in a dose-dependent manner. Epigallocatechin-3-O-gallate, one of these three Compounds, is a typical polyphenol Compound derived from tea leaves. Structurally related Compounds were synthesized and examined for their activity, revealing a hydroxyl group at A-ring position 5 as important for its activity. The present findings provide valuable insight for the development of natural product-derived therapeutics for fibrotic diseases, including idiopathic pulmonary fibrosis.     

Mechanical strengthening of Si cantilever by chemical KOH etching and its surface analysis by TEM and AFM

Mechanical strengthening of a Si cantilever by applying KOH wet etching was investigated. Two kinds of Si cantilever specimens having the different crystallographic orientations of the sidewall surfaces, i.e., Si{100} and Si{110}, were fabricated from the same SOI wafer by a Bosch process. The typical height and pitch of the scalloping formed on the sidewall were 248 and 917 nm, respectively. A 50 % KOH (40 °C) chemical wet etching was applied to increase the fracture stress of the Si cantilever. The fracture stress in the both of Si{100} and Si{110} cantilevers increased with the advance of the etching. The obtained maximum fracture stress in Si{100} and Si{110} were 4.2 and 3.7 GPa, respectively. Sidewall surface of the cantilever was analyzed to investigate the mechanical strengthening of Si cantilever by wet etching. The etched surface crystalline was analyzed by the transmission electron microscope (TEM), and confirmed that the thickness of the affected flow layer was less than 10 nm from the obtained TEM image. Then the change of the surface roughness by the KOH etching was analyzed by the atomic force microscope. The surface was smoothened with the advance of the KOH etching. The roughness value of Ra in Si{100} and Si{110} decreased to 12.1 and 37.7 nm, respectively.     

Development of an epileptic seizure prediction algorithm using R–R intervals with self-attentive autoencoder

Epilepsy is a neurological disorder that may affect the autonomic nervous system (ANS) from 15 to 20 min before seizure onset, and disturbances of ANS affect R–R intervals (RRI) on an electrocardiogram (ECG). This study aims to develop a machine learning algorithm for predicting focal epileptic seizures by monitoring R–R interval (RRI) data in real time. The developed algorithm adopts a self-attentive autoencoder (SA-AE), which is a neural network for time-series data. The results of applying the developed seizure prediction algorithm to clinical data demonstrated that it functioned well in most patients; however, false positives (FPs) occurred in specific participants. In a future work, we will investigate the causes of FPs and optimize the developing seizure prediction algorithm to further improve performance using newly added clinical data.

     

The effect of small dose of topiroxostat on serum uric acid in patients receiving hemodialysis

Introduction : Topiroxostat, a recently developed xanthine oxidase inhibitor, is expected to have fewer adverse effects than allopurinol because it has different mechanism of action from alloprinol. However, its dosage, usage and safety have not been established in patients with impaired renal function or those undergoing dialysis at the development since no studies was conducted in these patients. Methods : Cross over clinical trial using 3 months of allopurinol and topiroxostat on 27 maintain Japanese HD patients were carried out. The effects on oxidative stress status of both drugs were also evaluated by measuring oxidation reduction potential. Findings : Twenty‐five of twenty‐seven patients completed study. The mean serum uric acid levels in the topiroxostat‐treated arm was significantly lower than it in the allopurinol‐treated arm time‐dependently (P < 0.0001). Corrected oxidative stress ratio defined as biological antioxidant potential/diacron reactive oxygen metabolites was significantly increased in topiroxostat‐arm (*P = 0.0035), but not in allopurinol‐arm (P = 0.1429). No significant difference was seen in diacron reactive oxygen metabolites, biological antioxidant potential, static oxidation‐reduction potential, and capacity oxidation‐reduction potential between pre and post treatment of both drugs. Discussion : It is suggested that a low dose of topiroxostat decreased serum uric acid sufficiently to maintain it below 7.0 mg/dL in patients receiving hemodialysis.     

Experimental measurement on tangential momentum accommodation coefficient in a single microtube

The tangential momentum accommodation coefficient (TMAC) was investigated experimentally from the mass flow rate through a single microtube under the slip flow and the early part of the transition regime. The measurements were carried out by the constant-volume method under the mean Knudsen number smaller than 0.3, which is based on the mean pressure of the inlet and the outlet of the microtube, to apply the second-order slip boundary condition. To measure TMACs on various materials, quite large microtube was employed, which require the reduction in leakage. TMAC was obtained from the slip coefficient determined by the relation of the mass flow rate to the mean Knudsen number. The obtained mass flow rate was well explained by the theoretical equation. TMACs of deactivated-fused silica with argon, nitrogen, and oxygen were measured, showing the tangential momentum was not accommodated completely to the surface, and the values showed good agreement with previous studies. From the comparison between microtubes with different inner diameter, it is showed that TMAC is determined mainly by gas species and surface material.     

Multidimensional Cube Packing

We consider the d-dimensional cube packing problem (d-CPP): given a list L of d-dimensional cubes and (an unlimited quantity of) d-dimensional unit-capacity cubes, called bins, find a packing of L into the minimum number of bins. We present two approximation algorithms for d-CPP, for fixed d. The first algorithm has an asymptotic performance bound that can be made arbitrarily close to 2 – (1/2)^d . The second algorithm is an improvement of the first and has an asymptotic performance bound that can be made arbitrarily close to 2 – (2/3)^d . To our knowledge, these results improve the bounds known so far for d = 2 and d = 3, and are the first results with bounds that are not exponential in the dimension.