Mechanically Robust Self-Organized Crack-Free Nanocellular Graphene with Outstanding Electrochemical Properties in Sodium Ion Battery.

Crack-free nanocellular graphenes are attractive materials with extraordinary mechanical and electrochemical properties, but their homogeneous synthesis on the centimeter scale is challenging. Here, a strong nanocellular graphene film achieved by the self-organization of carbon atoms using liquid metal dealloying and employing a defect-free amorphous precursor is reported. This study demonstrates that a Bi melt strongly catalyzes the self-structuring of graphene layers at low processing temperatures. The robust nanoarchitectured graphene displays a high-genus seamless framework and exhibits remarkable tensile strength (34.8 MPa) and high electrical conductivity (1.6 × 104 S m-1). This unique material has excellent potential for flexible and high-rate sodium-ion battery applications.

DKK3/CKAP4 axis is associated with advanced stage and poorer prognosis in oral cancer.

OBJECTIVE: We previously reported that dickkopf WNT signaling inhibitor 3 (DKK3) would modulate malignant potential of oral squamous cell carcinoma (OSCC) via activating Akt. Recently, cytoskeleton associated protein 4 (CKAP4) functions as receptor of DKK3, which activates Akt in esophageal squamous cell carcinoma, but its expression and function in OSCC were unclear. METHODS: We studied DKK3 and CKAP4 protein expression in OSCC tissue and investigated the correlation between protein expression and clinical data. We also investigated whether antibodies (Ab) for DKK3 or CKAP4 could suppress malignant potential of the cancer cells. RESULTS: DKK3/CKAP4 protein expression was observed in majority of OSCC cases and was associated with significantly higher T-stage and TNM stage. Multivariate analysis revealed that DKK3 and CKAP4 were independent prognostic biomarkers for overall survival (OS) and disease-free survival (DFS), respectively. Survival analyses revealed that DKK3-positive cases and CKAP4-positive cases showed significantly shorter OS and DFS, respectively, and that DKK3/CKAP4 double-negative cases showed significantly favorable prognosis. Both anti-DKK3Ab and anti-CKAP4Ab could suppress cancer cell proliferation, migration, and invasion. CONCLUSION: DKK3/CKAP4 axis is thought to be important in OSCC, and it would be a promising therapeutic target.

Antiparkinsonian drugs as potent contributors to nocturnal sleep in patients with Parkinson's disease.

OBJECTIVE: To clarify whether antiparkinsonian drugs contribute to nocturnal sleep disturbances in patients with Parkinson's disease (PD). BACKGROUND: Although the major antiparkinsonian drugs L-dopa and dopamine agonists (DAs) have been found to affect sleep, little is known about the effects of specific drugs on sleep in PD patients. METHODS: The study participants consisted of 112 PD patients (median age 72.5 years [inter-quartile range: IQR 65-79]; mean disease duration 8.44 years [standard deviation: 7.33]; median Hoehn and Yahr stage 3 [IQR 2-3.75]) taking one of three types of non-ergot extended-release DAs (rotigotine 32; pramipexole 44; ropinirole 36) with or without L-dopa (median daily total dosage of antiparkinsonian drugs 525.5 mg [IQR 376.25-658] levodopa equivalent dose [LED]). Participants were assessed using the PD Sleep Scale-2 (PDSS-2). RESULTS: For the whole PD patient cohort, the PDSS-2 sleep disturbance domain score and the scores for item 1 assessing sleep quality and item 8 assessing nocturia were positively correlated with daily total dosage of antiparkinsonian drugs and dosage of L-dopa, but not with the dosage of DAs. Sub-analysis according to DA treatment revealed that DA dosage was not correlated with item 1 or 8 score in any of the subgroups. The LED ratio of rotigotine to the total dosage of antiparkinsonian drugs was inversely correlated with the item 1 score. CONCLUSIONS: These data suggest that antiparkinsonian drugs, in particular L-dopa, adversely affect nocturnal sleep in PD patients, especially in terms of sleep quality and nocturia. Thus, adjusting both the total dosage of antiparkinsonian drugs and the dose-ratio of L-dopa might be key actions for alleviating poor sleep quality in patients with PD. Among DAs, we found a clear positive correlation between the dose-ratio of rotigotine and sleep quality. Thus, partial L-dopa replacement with rotigotine could improve sleep quality in patients with PD.

Austenite grain growth simulation considering the solute-drag effect and pinning effect.

The pinning effect is useful for restraining austenite grain growth in low alloy steel and improving heat affected zone toughness in welded joints. We propose a new calculation model for predicting austenite grain growth behavior. The model is mainly comprised of two theories: the solute-drag effect and the pinning effect of TiN precipitates. The calculation of the solute-drag effect is based on the hypothesis that the width of each austenite grain boundary is constant and that the element content maintains equilibrium segregation at the austenite grain boundaries. We used Hillert's law under the assumption that the austenite grain boundary phase is a liquid so that we could estimate the equilibrium solute concentration at the austenite grain boundaries. The equilibrium solute concentration was calculated using the Thermo-Calc software. Pinning effect was estimated by Nishizawa's equation. The calculated austenite grain growth at 1473-1673 K showed excellent correspondence with the experimental results.

[A case of hepatic cryptococcosis complicated by adult T-cell leukemia].

A woman in her 80s was admitted to our hospital on account of jaundice, abnormal liver function tests, and leukocytosis. She was diagnosed with adult T-cell leukemia on the basis of the presence of anti-human T-cell leukemia virus type I (HTLV-I) and the results of flow cytometric analysis of peripheral blood. She also showed lung consolidation and cavitation, and a sputum smear and culture revealed cryptococcal infection. Therefore, she was diagnosed with pulmonary cryptococcosis. However, the cause of the abnormal liver function tests and jaundice remained unclear, and the patient subsequently died. On autopsy, multiple granulomas were observed throughout the liver, consistent with cryptococcal bodies. Herein we report this rare case of hepatic cryptococcosis with predominant hepatobiliary complaints.

Clinical outcome of fragment fixation for osteochondritis dissecans of the elbow.

BACKGROUND: The choice of surgical or non-surgical treatments for osteochondritis dissecans (OCD) of the humeral capitellum is still controversial. The purpose of this study was to assess the efficacy of fragment fixation for OCD of the humeral capitellum. METHODS: We reviewed 28 patients with OCD of the humeral capitellum after a mean follow up of 17 months. All patients were men and mean age was 14 years. Twenty-seven patients had a history of repetitive overuse of the elbow with baseball pitching, one with tennis. Mean duration of overuse of the elbow was four years. All patients had elbow pain and difficulty in throwing, with a mean duration of symptoms for 17 months. The mean arc of flexion before surgery ranged from 11 degrees to 126 degrees. Radiographs of the elbow showed a radiolucent cystic area of the humeral capitellum in one patient, a non-displaced split type fragment in 12 patients, and a slightly displaced split type fragment in 15 patients. Fragment fixation surgery was performed in all patients by lateral arthrotomy including drilling and fixation of the fragment with a double wiring technique using flexible wire or thread under direct vision. Sport activities using upper extremities were restricted for four to six months until the lesion healed in radiograph. RESULTS: Post-operatively, 25 patients had no pain and three decreased pain. Average arc of flexion was one to 132 degrees, an improvement of 16 degrees compared with the pre-operative arc. Radiographic findings showed complete healing of the lesion in 11 patients, partial healing in 12, unchanged in three, and loose body formation in two. By Tivnon's evaluation of the elbow function, results were excellent in 19 patients, good in five, fair in two, and poor in two. The ratio of complete or partial healing of the lesion was 100 percent in 16 patients in whom the thickness of the lesion was less than 9 mm on pre-operative radiograph, and 58 percent in 12 patients in whom the lesion thickness was 9 mm or more, which showed a significant difference (p<0.01). CONCLUSIONS: Fragment fixation for OCD of the humeral capitellum was effective in patients whose lesion thickness was less than 9 mm. Fixation by flexible wire or thread and revascularization by drilling for the fragment were considered to be insufficient for large lesions with a thickness of 9 mm or more.

Down-regulation of heme oxygenase-2 is associated with the increased expression of heme oxygenase-1 in human cell lines.

Intracellular heme concentrations are maintained in part by heme degradation, which is catalyzed by heme oxygenase. Heme oxygenase consists of two structurally related isozymes, HO-1 and HO-2. Recent studies have identified HO-2 as a potential oxygen sensor. To gain further insights into the regulatory role of HO-2 in heme homeostasis, we analyzed the expression profiles of HO-2 and the biochemical consequences of HO-2 knockdown with specific short interfering RNA (siRNA) in human cells. Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and HepG2 hepatoma. Down-regulation of HO-2 expression with siRNA against HO-2 (siHO-2) caused induction of HO-1 expression at both mRNA and protein levels in HeLa and HepG2 cells. In contrast, knockdown of HO-1 expression did not noticeably influence HO-2 expression. HO-2 knockdown prolonged the half-life of HO-1 mRNA twofold in HeLa cells. Transient transfection assays in HeLa cells revealed that the 4.5-kb human HO-1 gene promoter was activated with selective knockdown of HO-2 in a sequence-dependent manner. Moreover, HO-2 knockdown caused heme accumulation in HeLa and HepG2 cells only when exposed to exogenous hemin. HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellular heme homeostasis. These results suggest that HO-2 may down-regulate the expression of HO-1, thereby directing the co-ordinated expression of HO-1 and HO-2.

Hypoxia reduces the expression of heme oxygenase-2 in various types of human cell lines. A possible strategy for the maintenance of intracellular heme level.

Heme oxygenase consists of two structurally related isozymes, heme oxygenase-1 and and heme oxygenase-2, each of which cleaves heme to form biliverdin, iron and carbon monoxide. Expression of heme oxygenase-1 is increased or decreased depending on cellular microenvironments, whereas little is known about the regulation of heme oxygenase-2 expression. Here we show that hypoxia (1% oxygen) reduces the expression levels of heme oxygenase-2 mRNA and protein after 48 h of incubation in human cell lines, including Jurkat T-lymphocytes, YN-1 and K562 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma, as judged by northern blot and western blot analyses. In contrast, the expression level of heme oxygenase-1 mRNA varies under hypoxia, depending on the cell line; it was increased in YN-1 cells, decreased in HeLa and HepG2 cells, and remained undetectable in Jurkat and K562 cells. Moreover, heme oxygenase-1 protein was decreased in YN-1 cells under the conditions used, despite the induction of heme oxygenase-1 mRNA under hypoxia. The heme oxygenase activity was significantly decreased in YN-1, K562 and HepG2 cells after 48 h of hypoxia. To explore the mechanism for the hypoxia-mediated reduction of heme oxygenase-2 expression, we showed that hypoxia shortened the half-life of heme oxygenase-2 mRNA (from 12 h to 6 h) in YN-1 cells, without affecting the half-life of heme oxygenase-1 mRNA (9.5 h). Importantly, the heme contents were increased in YN-1, HepG2 and HeLa cells after 48 h of incubation under hypoxia. Thus, the reduced expression of heme oxygenase-2 may represent an important adaptation to hypoxia in certain cell types, which may contribute to the maintenance of the intracellular heme level.

Hypoxemia and blunted hypoxic ventilatory responses in mice lacking heme oxygenase-2.

Heme oxygenase (HO) catalyzes physiological heme degradation and consists of two structurally related isozymes, HO-1 and HO-2. Here we show that HO-2-deficient (HO-2(-/-)) mice exhibit hypoxemia and hypertrophy of the pulmonary venous myocardium associated with increased expression of HO-1. The hypertrophied venous myocardium may reflect adaptation to persistent hypoxemia. HO-2(-/-) mice also show attenuated ventilatory responses to hypoxia (10% O2) with normal responses to hypercapnia (10% CO2), suggesting the impaired oxygen sensing. Importantly, HO-2(-/-) mice exhibit normal breathing patterns with normal arterial CO2 tension and retain the intact alveolar architecture, thereby excluding hypoventilation and shunting as causes of hypoxemia. Instead, ventilation-perfusion mismatch is a likely cause of hypoxemia, which may be due to partial impairment of the lung chemoreception probably at pulmonary artery smooth muscle cells. We therefore propose that HO-2 is involved in oxygen sensing and responsible for the ventilation-perfusion matching that optimizes oxygenation of pulmonary blood.

Erythropoietin regulates tumour growth of human malignancies.

In addition to the chief function of erythropoietin (Epo) in promoting erythropoiesis, some other roles have been found in the brain and uterus. We have reported that signalling pathways of Epo and Epo receptor (EpoR) are involved in the tumourigenesis of ovarian and uterine cancers. To determine whether Epo plays a similar role in other malignancies, we studied the expression of Epo in several malignant human cell lines. We found that 24 malignant human cell lines examined express Epo and EpoR regardless of their origins, types, genetic characteristics and biological properties and secrete a very small amount of Epo individually and that most of them respond to hypoxic stimuli by enhanced secretion of Epo. To determine whether the Epo-EpoR pathway operates in tumours of these cell lines, we transplanted several cell lines into nude mice and confirmed the presence of Epo-responsive sites in xenografts in which the phosphorylation of the STAT5 (signal transducer and activator of transcription) is detectable. Furthermore, in nude mice we blocked the Epo signalling in xenografts of two representative cell lines, stomach choriocarcinoma and melanoma, by i.p. injections of EpoR antagonist and found inhibition of angiogenesis and survival of tumour cells leading to destruction of tumour masses and disturbances of phosphorylation of STAT5. In contrast, Epo mimetic peptide promotes angiogenesis and tumour cell survival. These findings suggest that Epo is indispensable for the growth and viability of malignant tumour and also that the deprivation of Epo signalling may be a promising therapy for human malignancy.

Bach1 functions as a hypoxia-inducible repressor for the heme oxygenase-1 gene in human cells.

Heme oxygenase 1 (HO-1) catalyzes heme breakdown, eventually releasing iron, carbon monoxide, and bilirubin IXalpha. HO-1 is induced by its substrate heme and various environmental factors, which represents a protective response against oxidative stresses. Here we show that hypoxia represses HO-1 expression in three human cell types but induces it in rat, bovine, and monkey cells, indicating the inter-species difference in the hypoxic regulation of HO-1 expression. The hypoxia-mediated repression of HO-1 expression is consistently associated with the induction of Bach1, a heme-regulated transcriptional repressor, in human cells. Bach1 is a basic leucine zipper protein, forming a heterodimer with a small Maf protein. Expression of HO-1 was also reduced in human cells when exposed to interferon-gamma or an iron chelator desferrioxamine, each of which induced Bach1 expression. In contrast, induction of HO-1 expression by CoCl(2) is associated with reduced expression of Bach1 mRNA. Thus, expression of HO-1 and Bach1 is inversely regulated. We have identified a Maf recognition element in the human HO-1 gene that is required for repression of a reporter gene by hypoxia and targeted by Bach1. Therefore, Bach1 functions as a hypoxia-inducible repressor for the HO-1 gene, thereby contributing to fine-tuning of oxygen homeostasis in human cells.

Lead, chemical porphyria, and heme as a biological mediator.

One of the most well-characterized symptoms of lead poisoning is porphyria. The biochemical signs of lead intoxication related to porphyria are delta-aminolevulinic aciduria, coproporphyrinuria, and accumulation of free and zinc protoporphyrin in erythrocytes. From the 1970s to the early 80s, almost all of the enzymes in the heme pathway had been purified and characterized, and it was demonstrated that delta-aminolevulinic aciduria is due to inhibition of delta-aminolevulinate dehydratase by lead. Lead also inhibits purified ferrochelatase; however, the magnitude of inhibition was essentially nil even under pathological conditions. Further study proved the disturbance of iron-reducing activity by moderate lead exposure. Far different from these two enzymes, lead failed to inhibit purified coproporphyrinogen oxidase, i.e., the mechanism of coproporphyrinuria has not yet been understood. During the 80s to the 90s, the effects of environmental hazards including lead were elucidated through stress proteins, indicating the induction of some heme pathway enzymes as stress proteins. At that time, gene environment interaction was another focus of toxicology, since gene carriers of porphyrias are considered to be a high-risk group to chemical pollutants. Toxicological studies from the 70s to the 90s focused on the direct effect of hazards on biological molecules, such as the heme pathway enzymes, and many environmental pollutants were proved to affect cytosolic heme. Recently, we demonstrated the mechanism of the heme-controlled transcription system, which suggests that the indirect effects of environmental hazards are also important for elucidating toxicity, i.e., the hazards can affect cell functions through such biological mediators as regulatory heme. It is, therefore, probable that toxicology in the future will focus on biological systems such as gene regulation and signal transduction systems.

[Heme metabolism in stress response].

Heme and its metabolism fulfill significant roles in many homeostatic and adaptative reactions. For example, heme (protein) senses oxygen concentration to regulate hypoxia response genes such as erythropoietin, and free heme, a proxidant, controls levels of several oxidative stress response proteins as well as that of a few enzymes in the heme metabolic pathway. Heme oxygenase (HO) is the key enzyme in heme catabolism, which degrades heme to Fe, CO, and biliverdin. CO is known as a gaseous messenger in the vascular and nervous systems. Biliverdin is rapidly converted to bilirubin, whose antioxidative effect is proposed to protect cells against reactive oxygen species. HO-1, the inducible isozyme of HO, is induced not only by its substrate heme, but also NO, metals, hypoxia, and various other stimuli. Studies on HO-1 deficiency indicate that induction of HO-1 is essential to homeostasis, at least in humans. Heme response elements (HREs), which mediate the induction of HO-1 expression by heme, are identified in enhancer regions of the mouse HO-1 gene. HRE shares a nucleotide sequence with the Maf recognition element (MARE). A transcriptional activator, Nrf2, has been shown to participate in HO-1 induction by several stimuli, including heme via HRE. A heme-binding transcription factor such as yeast Hap1 had been supposed to also exist in vertebrates, however, no such factor had been identified. Recently, we found that a mammalian transcription repressor, Bach1, directly binds heme, and that the DNA binding activity of Bach1 is negatively regulated by heme. Bach1 is capable of competing the binding to MARE with activators including Nrf2, therefore, HO-1 and other stress response genes bearing MARE may be regulated by heme via the MARE-binding transcription factors. Further analyses on the gene regulatory mechanism by heme would help us to understand the stress response system, especially against oxidative stress.

Regulatory heme and trichloroethylene intoxication: A possible explanation of the case of "A Civil Action".

In 1998, a amovie entitled "A Civil Action" was released. The movie described the Woburn case, begun in 1982 and concluded in 1990, one of the most famous cases of trichloroethylene pollution. In a small town near Boston, twelve children died of leukemia, which seemed attributable to trichloroethylene contamination of the drinking water. The victims, however, could not win the case, since evidence that the identified chemicals could cause leukemia and other human illnesses was rather sketchy. There have been many cases of trichloroethylene pollution in industrial nations including Japan, therefore, we reconsidered the missing link. Our conclusion is that the disease occurred not by a direct effect of the chemical hazard on biological macromolecules but by an indirect effect through the physiological system such as signal transduction and transcriptional regulation. In 1984, we reported a marked reduction in the regulatory heme pool by trichloroethylene exposure, however, the biological significance was not well understood. Recently, we found that the DNA binding activity of Bach1, a negative regulator of genes, is controlled by heme, the regulation of which seems to explain how leukemia develops. The heterodimer of Bach1 with MafK recognizes Maf recognition elements (MAREs) competing with the erythroid type positive regulator, a complex of NF-E2 with MafK. Bach1/MafK occupies MAREs under lower heme conditions, whereas MAREs are open to NF-E2/MafK along with increasing heme concentration. Since the NF-E2/MafK function is closely related to normal erythroid differentiation, of which disorders such as sideroblastic anemia are often related to neoplasia; i.e., a clonal disorder that can progress to leukemia. Thus, a marked decline in regulatory heme by trichloroethylene intoxication could be one of the pathways to leukemia.