Loss of SDHB Elevates Catecholamine Synthesis and Secretion Depending on ROS Production and HIF Stabilization.

Germline mutations in genes encoding succinate dehydrogenase subunits are associated with the development of familial pheochromocytomas and paragangliomas [hereditary paraganglioma/pheochromocytoma syndrome (HPPS)]. In particular, a mutation in succinate dehydrogenase subunit B (SDHB) is highly associated with abdominal paraganglioma and subsequent distant metastasis (malignant paraganglioma), indicating the importance of SDHB genetic testing. The discovery of HPPS suggests an association among genetic mitochondrial defects, tumor development, and catecholamine oversecretion. To investigate this association, we transfected pheochromocytoma cells (PC12) with SDHB-specific siRNA. SDHB silencing virtually abolished complex II activity, demonstrating the utility of this in vitro model for investigating the pseudo-hypoxic drive hypothesis. Lack of complex II activity resulting from RNA interference of SDHB increased tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamine biosynthesis) activity and catecholamine secretion. Reduced apoptosis was observed accompanied by Bcl-2 accumulation in PC12 cells, consistent with the phenotypes of paragangliomas with SDHB mutations. In addition, SDHB silencing increased reactive oxygen species (ROS) production and nuclear HIF1α stabilization under normoxic conditions. Furthermore, phenotypes induced by complex II activity knockdown were abolished by pretreatment with N-acetyl cysteine (an ROS scavenger) and by prior HIF1α knockdown, indicating an ROS- and HIF1α-dependent mechanism. Our results indicate that increased ROS may act as signal transduction messengers that induce HIF1α stabilization and may be necessary for the pseudo-hypoxic states observed in our experimental model. To our knowledge, this is the first study demonstrating that pseudo-hypoxic states resulting from SDHB knockdown are associated with increased TH activity and catecholamine oversecretion.

Identification and formation mechanism of individual degradation products in lithium-ion batteries studied by liquid chromatography/electrospray ionization mass spectrometry and atmospheric solid analysis probe mass spectrometry.

RATIONALE: Improvement of lithium ion batteries (LIBs) in terms of performance and robustness requires good understanding of the reaction processes. The analysis of the individual degradation products in LIB electrolytes and on the surface of the electrodes provides vital information in this regard. In this study, mass spectrometric analytical methods were utilized for the identification of the individual degradation products. METHODS: The degradation products in the electrolytes recovered from cycle-tested cells were separated by liquid chromatography (LC) and their mass spectrometric analysis was conducted by electrospray ionization mass spectrometry (ESI-MS). For identification of degradation products on the surface of electrodes, atmospheric solid analysis probe (ASAP)-MS analysis was conducted by time-of-flight mass spectrometry with an ASAP probe and an atmospheric pressure chemical ionization source. RESULTS: The degradation products in the electrolytes, namely carbonate oligomers and organophosphates, were identified simultaneously by LC/ESI-MS. Their formation mechanisms were estimated, which explain their different compositions at different temperatures. One degradation product was found on the anode surface by ASAP-MS, and its formation mechanism was explained similarly to those in the electrolyte. CONCLUSIONS: The results suggest that the electrolyte degradation is correlated with the formation of a solid electrolyte interphase, which is an important factor in the performance of LIBs. We expect that further investigation of the degradation products by LC/ESI-MS and ASAP-MS will be helpful for studying their degradation processes in LIBs. Copyright © 2016 John Wiley & Sons, Ltd.

Inhibition of autophagy enhances sunitinib-induced cytotoxicity in rat pheochromocytoma PC12 cells.

Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors, and recently, it has been shown to be an active agent for the treatment of malignant pheochromocytomas. Previously, we demonstrated that sunitinib directly inhibited mTORC1 signaling in rat pheochromocytoma PC12 cells. Although autophagy is a highly regulated cellular process, its relevance to cancer seems to be complicated. It is of note that inhibition of mTORC1 is a prerequisite for autophagy induction. Indeed, direct mTORC1 inhibition initiates ULK1/2 autophosphorylation and subsequent Atg13 and FIP200 phosphorylation, inducing autophagy. Here, we demonstrated that sunitinib significantly increased the levels of LC3-II, concomitant with a decrease of p62 in PC12 cells. Following sunitinib treatment, immunofluorescent imaging revealed a marked increased punctate LC3-II distribution. Furthermore, Atg13 knockdown significantly reduced its protein level, which in turn abolished sunitinib-induced autophagy. Moreover, inhibition of autophagy by siRNAs targeting Atg13 or by pharmacological inhibition with ammonium chloride, enhanced both sunitinib-induced apoptosis and anti-proliferation. Thus, sunitinib-induced autophagy is dependent on the suppression of mTORC1 signaling and the formation of ULK1/2-Atg13-FIP200 complexes. Inhibition of autophagy may be a promising therapeutic option for improving the anti-tumor effect of sunitinib.

Evaluation of 95-Gene Classifier of Formalin-fixed Paraffin-embedded Tissues in ER-positive, HER2-negative, and Node-negative Breast Cancer.

BACKGROUND/AIM: A subset of patients with estrogen receptor (ER)-positive, HER2-negative, and node-negative breast cancer experience recurrences. Predicting patients who will have recurrences within 5 years of surgery is essential so that patients can be selected to receive adjuvant chemotherapy. The 95-gene classifier (95-GC) has been validated as a method to differentiate patients into high and low-risk groups for early recurrence. PATIENTS AND METHODS: In this study, we performed 95-GC analysis on 56 formalin-fixed paraffin-embedded (FFPE) tissue samples from patients who underwent surgery for ER-positive, HER2-negative, and node-negative breast cancer and did not receive adjuvant chemotherapy. We associated the obtained high- and low-risk groups with clinicopathological characteristics and recurrence-free survival (RFS). RESULTS: We classified 12 out of 56 patients into the high-risk recurrence group. We found significantly higher KI67 scores in patients in the high-risk group. Other clinicopathological characteristics were not associated with the 95-GC risk groups. Patients in the 95-GC low-risk group had a significantly better prognosis than those in the high-risk group (p=0.0387). The 5-year RFS rate was 97.6% in the low-risk group and 74.1% in the high-risk group, while the 10-year RFS rates were 90.1% and 74.1%, respectively. CONCLUSION: The 95-GC score can accurately predict RFS within 5 years of surgery for ER-positive, HER2-negative, and node-negative breast cancer using FFPE tissue samples. These prediction models could help assign patients to the most effective treatment regimen.

Sex-inducing effects toward planarians widely present among parasitic flatworms.

Various parasitic flatworms infect vertebrates for sexual reproduction, often causing devastating diseases in their hosts. Consequently, flatworms are of great socioeconomic and biomedical importance. Although the cessation of parasitic flatworm sexual reproduction is a major target of anti-parasitic drug design, little is known regarding bioactive compounds controlling flatworm sexual maturation. Using the planarian Dugesia ryukyuensis, we observed that sex-inducing substances found in planarians are also widespread in parasitic flatworms, such as monogeneans and flukes (but not in tapeworms). Reverse-phase HPLC analysis revealed the sex-inducing substance(s) eluting around the tryptophan retention time in the fluke Calicophoron calicophorum, consistent with previous studies on the planarian Bipalium nobile, suggesting that the substance(s) is likely conserved among flatworms. Moreover, six of the 18 ovary-inducing substances identified via transcriptome and metabolome analyses are involved in purine metabolism. Our findings provide a basis for understanding and modifying the life cycles of various parasitic flatworms.

Sunitinib induces apoptosis in pheochromocytoma tumor cells by inhibiting VEGFR2/Akt/mTOR/S6K1 pathways through modulation of Bcl-2 and BAD.

Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors (VEGFRs). Very recently, sunitinib has been shown to be an active agent for the treatment of malignant pheochromocytomas. However, it is unclear whether sunitinib acts only through an antiangiogenic mechanism or whether it may also directly target tumor cells. Sunitinib markedly induced apoptosis of PC12 cells in a dose-dependent and time-dependent manner. Furthermore, in support of these findings, we found that sunitinib induced a reduction in the expression of the antiapoptotic molecule Bcl-2 as well as dephosphorylation of the proapoptotic molecule BAD, which results in the activation of BAD in these cells. Consistent with these apoptotic effects, our results showed that sunitinib inhibited phosphorylation of Akt and mTOR and was followed by a reduction of S6K1, which is a well-known target of mTOR. Knockdown of VEGFR-2 attenuated the sunitinib-induced effects, including apoptosis and inhibition of signaling pathways such as the phosphorylation of Akt as well as mTOR, and Bcl-2, which confirmed that these effects could be mediated by VEGFR-2. In addition, silencing of S6K1 induced apoptosis accompanied by a decrease in the phosphorylation of BAD and Bcl-2, similar to that observed with sunitinib treatment. Thus, these results together suggest that sunitinib initially exerts its apoptotic effect through the inhibition of VEGFR-2, which, when followed by reduction of its downstream effectors, including Akt/mTOR/S6K1, may lead to inhibition of the antiapoptotic molecule Bcl-2 and activation of the proapoptotic molecule BAD in PC12 cells. However, PC12 cells do not precisely reflect the pathogenesis of malignant cells. Therefore, we confirmed the key findings by replicating these experiments in human neuroblastoma SK-N-SH cells.

Sunitinib inhibits catecholamine synthesis and secretion in pheochromocytoma tumor cells by blocking VEGF receptor 2 via PLC-γ-related pathways.

Sunitinib is an oral, small molecule multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that primarily targets vascular endothelial growth factor receptors (VEGFRs). Although sunitinib is an active agent for the treatment of malignant pheochromocytomas, it is unclear whether sunitinib acts through only antiangiogenic mechanisms or also directly targets tumor cells. We previously showed that sunitinib directly induced apoptosis of PC-12 cells. To further confirm these direct effects, we examined the effects of sunitinib on tyrosine hydroxylase (TH) (the rate-limiting enzyme in catecholamine biosynthesis) activity and catecholamine secretion in PC-12 cells and the underlying mechanisms. Sunitinib inhibited TH activity in a dose-dependent manner, and decreased TH protein levels. Consistent with this finding, sunitinib decreased TH phosphorylation at Ser(31) and Ser(40) and significantly decreased catecholamine secretion. VEGFR-2 knockdown attenuated these effects, including inhibition of TH activity and catecholamine secretion, suggesting that they were mediated by VEGFR-2. Sunitinib significantly decreased phospholipase C (PLC)-γ phosphorylation and subsequent protein kinase C (PKC) activity. Because Ser(40) phosphorylation significantly affects TH activity and is known to be regulated by PKC, sunitinib may inhibit Ser(40) phosphorylation via the VEGFR-2/PLC-γ/PKC pathway. Additionally, sunitinib markedly decreased the activity of extracellular signal-regulated kinase (ERK), but not c-Jun NH(2)-terminal kinase or p38 mitogen-activated protein kinase. Therefore, sunitinib may reduce TH Ser(31) phosphorylation through inhibition of the VEGFR-2/PLC-γ/PKC/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/ERK pathway. Sunitinib also significantly reduced inositol 1,4,5-trisphosphate production. However, because PC-12 cells do not precisely reflect the pathogenesis of malignant cells, we confirmed the key findings in a human neuroblastoma cell line, SK-N-SH. In conclusion, sunitinib directly inhibits catecholamine synthesis and secretion in pheochromocytoma PC-12 cells.

Identical germline mutations in the TMEM127 gene in two unrelated Japanese patients with bilateral pheochromocytoma.

OBJECTIVE: Recently, TMEM127 was shown to be a new pheochromocytoma susceptibility gene; this is consistent with its function as a tumour suppressor gene (Journal of Clinical Endocrinology and Metabolism, 2009, 94, 2817). Most pheochromocytomas arise from the adrenal medulla, and in approximately half of the cases, the tumours are bilateral (Journal of Clinical Endocrinology and Metabolism, 2009, 94, 2817; Journal of the American Medical Association, 2004, 292, 943; Human Mutation, 2010, 31, 41; Science, 2009, 325, 1139). The aim of the present study was to determine whether TMEM127 mutations are involved in the pathogenesis of pheochromocytomas/paragangliomas in Japanese subjects. PATIENTS AND METHODS: For this study, 74 unrelated patients with pheochromocytoma/paraganglioma who tested negative for mutations and deletions in RET, VHL, SDHB and SDHD were recruited through a multi-institutional collaborative effort in Japan. The TMEM127 gene sequence was determined in their germline DNA, and tumour DNA was analysed for the loss of heterozygosity. In addition, their TMEM127 gene sequences were compared with sequences from 114 normal healthy, ethnically matched controls. RESULTS: Among the 74 eligible patients, two unrelated patients (2·7%) with bilateral adrenal pheochromocytoma were found to have an identical germline TMEM127 mutation (c.116_119delTGTC, p.Ile41ArgfsX39) associated with 2q deletion loss of heterozygosity, which was also previously described in a Brazilian case (Journal of the American Medical Association, 2004, 292, 943). We also determined that none of the 114 normal healthy controls had this deletion mutation. CONCLUSION: This is the first report showing that TMEM127 mutation plays a pathological role in pheochromocytoma in an Asian population. Although our surveillance is limited, the prevalence and the phenotype of this gene mutation appear to be similar to those reported in previous studies.

Ion Transport in Solid Medium-Evaluation of Ionic Mobility for Design of Ion Transport Pathways in Separator and Gel Electrolyte.

Further improvement in the performance of lithium secondary batteries will be an indispensable issue to realize a decarbonized society. Among them, the batteries for electric vehicles still have many issues to be addressed because they are subject to various conditions such as high-power performance, safety, and cost restrictions for widespread use. Those subjects require extensive researches from the improvement of each element material to control the battery system to optimize the total performance. Based on this idea, we have been conducting research focusing on ion movement to elucidate the ion conduction mechanism from the microscopic point of view. It has been recognized that the ionic mobility in the battery, which dominates the power performance of the battery, is affected by the solid environment in which the ions move (separator and electrode materials) and the evaluation of ion movement, including the interaction with the surroundings, is necessary as an essential step for battery design. In this article, I will introduce the evaluation approach of ion dynamics and the evaluation results of mobility and interactive situations of carrier ions in the practical separator membranes and gel electrolytes. Finally, the direction of material design is outlined through this review.

Direct measurements of ionic mobility of ionic liquids using the electric field applying pulsed gradient spin-echo NMR.

Ionic mobilities of the ionic liquids, 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)amide,1-ethyl-3-methylimidazolium fluorosulfonyl-(trifluoromethylsulfonyl)amide, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl) -amide, were measured using the electric field applying pulsed gradient spin-echo NMR technique. Observed mobilities were more than 1 order of magnitude greater than the values estimated from the diffusion coefficients measured under the equilibrium state without the electric field. Electric field dependence of the ionic mobility showed that the high mobility appeared above the threshold of the field strength with keeping the constant values. This indicates that the ions are orientated by the application of the electric field may be due to the dielectric polarization.

Development of recurrence risk score using 95­gene classifier and its application to formalin­fixed paraffin­embedded tissues in ER­positive, HER2­negative and node­negative breast cancer.

We previously developed a 95­gene classifier (95GC) to classify ER­positive/HER2­negative/node­negative (ER+/HER2­/N0) breast cancer as high­ and low­risk. The present study aimed to devise a 95GC recurrence score (95GCRS) to estimate recurrence risk more precisely and, although the 95GC was originally developed using fresh­frozen (FF) tissues, this was applied to formalin­fixed paraffin­embedded (FFPE) tissues. 95GCRS was calculated using between­group analysis and denominated as a value from 0 to 100. Correlation of 95GCRS with distant recurrence rate and response to neoadjuvant chemotherapy (NAC) was evaluated in 257 patients with ER+/HER2-/N0 breast cancer treated with adjuvant hormonal therapy at Osaka University Hospital and in 425 patients with ER+ breast cancer treated with NAC at Osaka University Hospital and the University of Texas MD Anderson Cancer Center (GSE25066 dataset). Correlation of 95GCRS between FF and FFPE tissues was evaluated in paired tissues from 56 ER+/HER2­/N0 breast cancer types obtained from patients without NAC treatment. Distant recurrence rates were remarkably low in patients with 95GCRS ≤50 and increased proportionally in patients with 95GCRS >50. Pathological complete response (pCR) rates to NAC were increased in proportion to 95GCRS, indicating a greater sensitivity of breast cancers with high 95GCRS to chemotherapy. 95GCRS was highly correlated (R=0.92) between FF and FFPE tissues, and the concordance rate (94.6%) of high­ and low­risk groups was also considerably high. Overall, the present study developed a 95GCRS that correlated with distant recurrence rate and pCR rate to NAC. The 95GC was applicable to FFPE tissues with a high concordance rate in FF tissues.

Ionization condition of lithium ionic liquid electrolytes under the solvation effect of liquid and solid solvents.

Ionization condition and ionic structures of the lithium ionic liquid electrolytes, LiTFSI/EMI-TFSI/(PEG or silica), were investigated through the measurements of ionic conductivity and diffusion coefficient. The size of the hydrodynamic lithium species (rLi) evaluated from the Stokes-Einstein equation was 0.90 nm before gelation with the PEG or silica. This reveals that the TFSI- anions from the solvent are coordinated on Li+ for solvation, forming, for example, Li(TFSI)4(3-) and Li(TFSI)2- in the electrolyte solution. By the dispersion of PEG for gelation, rLi increased up to 1.8 nm with the 10 wt % of PEG. This indicates that the lithium species is directly interacted with the oxygen sites on the polymer chains and the lithium species migrate, reflecting the polymer by hopping from site to site. In case of the silica dispersion, rLi decreased to 0.7 nm at 10 wt % silica. Although the silica surface with silanol groups fundamentally attracts Li+, the lithium does not migrate from site to site on the silica surface as in the gel of the polymer and follows random walk behavior in the network of the liquid-phase pathways in the two-phase gel. In the process, that solvated TFSI- anions are partially removed may be due to the attractive effect of H+, which was dissociated from the silanol group. It is concluded that the dispersed silica is effective to modify the hydrodynamic lithium species to be appropriate for charge transport as reducing the size and anionic charge of Li(TFSI)4(3-) by removing one or two TFSI- anions.

Existing condition and migration property of ions in lithium electrolytes with ionic liquid solvent.

Ionization conditions of each ionic species in lithium ionic liquid electrolytes, LiTFSI/BMI-TFSI and LiTFSI/BDMI-TFSI, were confirmed based on the diffusion coefficients of the species measured by the pulsed gradient spin-echo (PGSE) NMR technique. We found that the diffusion coefficient ratios of the cation and anion species D(Li)(obs)/D(F)(obs) of the lithium salt and D(H)(obs)/D(F)(obs) of the ionic liquid solvent were effective guides to evaluate the ionization condition responsible for their mobility. Lithium ions were found to be stabilized, forming the solvated species as Li(TFSI)3(2-). TFSI- anion coordination could be relaxed by the dispersion of silica to form a gel electrolyte, LiTFSI/BDMI-TFSI/silica. It is expected that the oxygen sites on the silica directly attract Li+, releasing the TFSI- coordination. The lithium species, loosing TFSI- anions, kept a random walk feature in the gel without the diffusion restriction attributed from the strong chemical and morphological effect as that in the gel with the polymer. We can conclude that the silica dispersion is a significant approach to provide the appropriate lithium ion condition as a charge-transporting species in the ionic liquid electrolytes.

Ionization state and ion migration mechanism of room temperature molten dialkylimidazolium fluorohydrogenates.

The ionization state of room temperature molten salts, alkylimidazolium fluorohydrogenates (RMIm(HF)(2.3)F: R = alkyl group, M =methyl group), was evaluated from the observed diffusion coefficient and viscosity, using the Stokes-Einstein relation. Assuming that the dissociation degree of the salt is acceptable for representation of the ionic state of the molten salts, the larger the cation size with elongation of the alkyl chain was, the higher the dissociation degree of the salt. Further, we proposed that an idea of the "degree of ordering of cations and anions" was more suitable to represent the ionization state without solvent species. On the basis of this idea, the smaller the cation size of RMIm(HF)(2.3)F salt was, the higher the ordering of the ion, indicating formation of domain particles of aggregated ions as a unit of mobile species such as A(AX)m+ and X(AX)n- for A+ X- salt. It was found that highly ordered particles, with large numbers for m and n, showed a high diffusion coefficient.

Ionic diffusion and salt dissociation conditions of lithium liquid crystal electrolytes.

Salt dissociation conditions and dynamic properties of ionic species in liquid crystal electrolytes of lithium were investigated by a combination of NMR spectra and diffusion coefficient estimations using the pulsed gradient spin-echo NMR techniques. Activation energies of diffusion (Ea) of ionic species changed with the phase transition of the electrolyte. That is, Ea of the nematic phase was lower than that of the isotropic phase. This indicates that the aligned liquid crystal molecules prepared efficient conduction pathways for migration of ionic species. The dissociation degree of the salt was lower compared with those of the conventional electrolyte solutions and polymer gel electrolytes. This is attributed to the low concentration of polar sites, which attract the dissolved salt and promote salt dissociation, on the liquid crystal molecules. Furthermore, motional restriction of the molecules due to high viscosity and molecular oriented configuration in the nematic phase caused inefficient attraction of the sites for the salt. With a decreased dissolved salt concentration of the liquid crystal electrolyte, salt dissociation proceeded, and two diffusion components attributed to the ion and ion pair were detected independently. This means that the exchange rate between the ion and the ion pair is fairly slow once the salt is dissociated in the liquid crystal electrolytes due to the low motility of the medium molecules that initiate salt dissociation.

Evaluation of interactive effects on the ionic conduction properties of polymer gel electrolytes.

Ionic mobility of electrolyte materials is essentially determined by the nanoscale interactions, the ion-ion interactions and ion-solvent interactions. We quantitatively evaluated the interactive situation of the lithium polymer gel electrolytes through the measurements of ionic conductivity and diffusion coefficients of the mobile species of the lithium polymer electrolytes. The interactive force between the cation and anion in the gel depended on the mixing ratio of the binary solvent, ethylene carbonate plus dimethyl carbonate (EC/DMC). The gel with the solvent (3:7 EC:DMC) showed minimal cation-anion interaction, which is the cause of the highest ionic mobility compared with those of the other gels with different solvents. This suggests that the cation-anion interaction does not simply depend on the dielectric constant of the solvent but is associated with the solvation condition of the lithium. In the case of the gel with the 3:7 EC/DMC solvent, most of the EC species strongly coordinate to a lithium ion, forming the stable solvated lithium, Li(EC)(3)(+), and there are no residual EC species for exchange with them. As a result, the solvating EC species would be a barrier that restricts the anion attack to the lithium leading to the smallest cation-anion interaction. On the other hand, interaction between the cation and polar sites, hydroxyl and oxygen groups of ether of the polyvinyl butyral (PVB) and polyethylene oxide (PEO) polymer, respectively, in the gels was another dominant factor responsible for cation mobility. It increased with increasing polar site concentration per lithium. In case of the PVB gels, cation-anion interaction increased with an increasing polymer fraction of the gel contrary to the independent feature of PEO gels with the change of the polymer fraction. This indicates that the cation-anion interaction is associated with the polymer structure of the gel characterized by the kind and configuration of polar groups, molecular weight, and network morphology of the polymer.

Alkoxy chains in ionic liquid anions; effect of introducing ether oxygen into perfluoroalkylborate on physical and thermal properties.

The melting point and viscosity of [CF(3)OCF(2)CF(2)BF(3)](-) based ionic liquids are significantly lower than those of [CF(3)CF(2)CF(2)CF(2)BF(3)](-) based ionic liquids, indicating that the oxygen atom plays a key role in the preparation of low-melting and low-viscosity ionic liquids.