Integrated mechanistic and data-driven modelling for multivariate analysis of signalling pathways.

Mathematical models of highly interconnected and multivariate signalling networks provide useful tools to understand these complex systems. However, effective approaches to extracting multivariate regulation information from these models are still lacking. In this study, we propose a data-driven modelling framework to analyse large-scale multivariate datasets generated from mathematical models. We used an ordinary differential equation based model for the Fas apoptotic pathway as an example. The first step in our approach was to cluster simulation outputs generated from models with varied protein initial concentrations. Subsequently, decision tree analysis was applied, in which we used protein concentrations to predict the simulation outcomes. Our results suggest that no single subset of proteins can determine the pathway behaviour. Instead, different subsets of proteins with different concentrations ranges can be important. We also used the resulting decision tree to identify the minimal number of perturbations needed to change pathway behaviours. In conclusion, our framework provides a novel approach to understand the multivariate dependencies among molecules in complex networks, and can potentially be used to identify combinatorial targets for therapeutic interventions.     

Development of Selective TGR5 Ligands Based on the 5,6,7,8-Tetrahydro-5,5,8,8-tetramethylnaphthalene Skeleton

TGR5, a G-protein-coupled receptor (GPCR), plays an important role in several physiological functions. TGR5 activation through bile acids induces an increase in energy expenditure. Therefore, synthetic TGR5 ligands could be useful for the treatment of obesity or dyslipidemia. In this study, we designed and synthesized a set of TGR5 ligands with a 5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalene (TMN) skeleton, and evaluated their TGR5 agonistic activity. We also investigated the selectivity of the synthesized compounds for TGR5 relative to the farnesoid X receptor (FXR) and retinoic acid receptor (RAR). Our results show that compound 4 b [N-(2-chlorophenyl)-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenecarboxamide] exhibited potent TGR5 agonist activity with an IC₅₀ value of 8.4 nM without significant cytotoxicity. In addition, compound 4 b showed only slight agonistic activity toward FXR and RAR at 1 μM treatment. These data indicate that compound 4 b is a selective TGR5 agonist, and could be a promising therapeutic agent for dyslipidemia.     

Molecular cloning of porcine growth differentiation factor 9 (GDF-9) cDNA and its role in early folliculogenesis: direct ovarian injection of GDF-9 gene fragments promotes early folliculogenesis

Growth differentiation factor-9 (GDF-9) is a growth factor secreted by oocytes in growing ovarian follicles. To investigate the ovarian function of GDF-9 in pigs, we first cloned porcine GDF-9 complementary DNA (cDNA), and then injected its gene fragments into the ovary in gilts. Porcine GDF-9 has open reading frame (ORF) homologies of 81.4%, 84.6%, 84.2%, 72.7% and 72.6% with its human, bovine, ovine, rat and mouse counterparts respectively. Regarding the deduced amino-acid sequence of the mature protein, the corresponding homologies reach 92.1%, 97.8%, 97.0%, 89.6% and 88.1% respectively. To investigate the role of GDF-9 in early folliculogenesis, the ovaries of 2-month-old prepubertal gilts were injected with GDF-9 gene fragments. The injection of porcine GDF-9 gene fragments resulted in an increase in the number of primary, secondary and tertiary follicles, concomitant with a decrease in the number of primordial follicles. These results indicated that exogenous GDF-9 can promote early folliculogenesis in the porcine ovary, and that a technique for direct ovarian injection of GFD-9 gene fragments may contribute to a novel therapy for prevention and treatment of infertility associated with ovarian dysfunction.     

Irradiation properties of T0 chopper components

We investigated the irradiation properties of the components of a T0 chopper. The organic materials in the rotor bearing grease, the magnetic fluids in seals, and the rubber in the timing belt, as well as the semiconductor materials in the rotation sensor and motor encoder, were all irradiated with high-energy γ-rays up to 100 kGy. No significant damage that shortens the lifetime of a T0 chopper was observed for the mechanical components. However, the semiconductor components were damaged by the irradiation. For the rotation sensor system detecting the rotor phase, the signal from a marker on the rotor shaft was transmitted outside the shielding by an optical fiber with radiation-proofing and the electrical circuits were removed from the beamline shielding. The lifetime of the motor encoder possibly meets the requirement for the maintenance period of the T0 chopper.     

Restoration of solid oxide fuel cell stacks after failure of partial cells

We investigated a solid oxide fuel cell stack that employs anode-supported planar cells in which two intermediate plates are installed every 10 cells to determine the influence of the separation and reconnection of the intermediate plates after high temperature operation. We showed that this separation and reconnection caused no significant degradation in stack performance. A 30-cell stack, which was constructed by removing two 10-cell sub-stacks from a 50-cell stack that had operated stably 1200 h, functioned well. The difference between the average voltages of the cells in the 50- and 30-cell stacks was less than 3% when the current density, fuel utilization, and oxygen utilization were 0.30 A cm⁻², 60%, and 15%, respectively. The 30-cell stack operated stably for about 1200 h with almost no degradation. These findings indicate that our stack can be restored after cells in the stack have broken down simply by removing the 10-cell sub-stacks that contain the broken cells and replacing them with undamaged 10-cell sub-stacks.     

Field emission monotron for THz emission

Small-signal negative conductance of a field emission monotron was calculated, which was considerably higher than that of a thermionic monotron due to strong bunching of emitted electrons in a field emission. The amplification constant of 28dB/mm is achieved at THz wave region by applying the monotron to a distributed amplifier and is sufficient to overcome power loss in a transmission line. In addition, a wide band frequency tunable electromagnetic wave source may be developed at THz wave region by providing a low loss microstrip line resonator with field emission array due to the gate voltage dependent phase constant of the line.     

The role of ATP-sensitive potassium channels in striatal dopamine release: an in vivo microdialysis study

Inactivation of the retinoblastoma (RB) gene is known to be implicated in the pathogenesis of several types of human cancers. Since structural alterations of the RB gene have not been well examined in human bladder cancer, we looked for mutations in the entire coding region of this gene using polymerase chain reaction (PCR) and single-strand conformational polymorphism analysis of RNA. We also examined allelic loss of the RB gene using PCR-based restriction fragment length polymorphism analysis. Of 30 samples obtained from patients with bladder cancer, eight (27%) were found to have RB gene mutations. DNA sequencing of the PCR products revealed five cases with single point mutations and three cases with small deletions. These mutations included one (10%) of ten low-grade (grade 1) tumours, four (50%) of eight intermediate-grade (grade 2) tumours and three (25%) of 12 high-grade (grade 3) tumours. Likewise, mutations were found in four (21%) of 19 superficial (pTa and pT1) tumours and four (36%) of 11 invasive (pT2 or greater) tumours. In 15 informative cases, loss of heterozygosity at the RB locus was shown in five cases (33%), three cases with RB mutations and two without them. These results suggest that RB gene mutations are involved in low-grade and superficial bladder cancers as well as in high-grade and invasive cancers.     

Phonon Dispersion Measurements of YBa2Cu3O6.15 and YBa2Cu3O6.95 by Time-of-Flight Neutron Spectroscopy

We measured the phonon dispersions of YBa₂Cu₃O_6.15 and YBa₂Cu₃O_6.95 by time-of-flight inelastic neutron scattering. The in-plane bond-stretching modes in the metallic phase showed a distinct a-b plane anisotropy beyond what is expected for structural origin. Such anisotropy in the longitudinal optical modes, which is absent in the TO, suggests strong in-plane anisotropy in the underlying electronic structure. Apical oxygen bond-stretching modes showed a large frequency change between the insulating and the metallic phases. This large softening also is beyond structural origin, and suggests the effect of local electronic environment.     

Purification and characterization of recombinant human granulocyte colony-stimulating factor (rhG-CSF) derivatives: KW-2228 and other derivatives

Various derivatives of recombinant human granulocyte colony-stimulating factor (rhG-CSF) have been overproduced in Escherichia coli with the strong, inducible trp promoter. A derivative designated as KW-2228 in which the amino acids were replaced at five positions showed more potent granulopoietic activity and stability than those of wild-type both in vitro and in vivo. The purification involved a sequential renaturation process and three-step chromatography. Refolding succeeded in very high yield using a urea system. The purity of KW-2228 was greater than 99% as measured by SDS-PAGE and HPLC analysis. According to circular dichroism and nuclear magnetic resonance spectroscopy, rhG-CSF and KW-2228 have very similar conformations. This suggests that the substitution of five amino acids does not appreciably change the conformation of hG-CSF. KW-2228 ([Ala1, Thr3, Tyr4, Arg5, and Ser17]-hG-CSF) and derivative A ([Ala1, Thr3, Tyr4, Arg5]-hG-CSF) are easily crystallized and they show similar in vitro activity. On the other hand, neither rhG-CSF nor derivative B ([Ser17]-hG-CSF) are crystallized under the same conditions. Thus, the four amino acid substitution (Ala1, Thr3, Tyr4, Arg5) of the N-terminal sequence may facilitate crystallization. The change of Cys17 to Ser may not influence the stability and activity of hG-CSF.