A low-density cDNA microarray with a unique reference RNA: pattern recognition analysis for IFN efficacy prediction to HCV as a model

We have designed and established a low-density (295 genes) cDNA microarray for the prediction of IFN efficacy in hepatitis C patients. To obtain a precise and consistent microarray data, we collected a data set from three spots for each gene (mRNA) and using three different scanning conditions. We also established an artificial reference RNA representing pseudo-inflammatory conditions from established hepatocyte cell lines supplemented with synthetic RNAs to 48 inflammatory genes. We also developed a novel algorithm that replaces the standard hierarchical-clustering method and allows handling of the large data set with ease. This algorithm utilizes a standard space database (SSDB) as a key scale to calculate the Mahalanobis distance (MD) from the center of gravity in the SSDB. We further utilized sMD (divided by parameter k: MD/k) to reduce MD number as a predictive value. The efficacy prediction of conventional IFN mono-therapy was 100% for non-responder (NR) vs. transient responder (TR)/sustained responder (SR) (P < 0.0005). Finally, we show that this method is acceptable for clinical application.     

Acylated flavonoids and phenol glycosides from Veronica thymoides subsp. pseudocinerea

A new acylated flavone glucoside, 3'-hydroxyscutellarein 7-O-(6'-O-protocatechuoyl)-beta-glucopyranoside (1), and a new phenol glucoside, 3,5-dihydroxyphenethyl alcohol 3-O-beta-glucopyranoside (6) were isolated from Veronica thymoides subsp. pseudocinerea together with seven known flavone, phenol and lignan glycosides; 3'-hydroxyscutellarein 7-O-(6'-O-trans-feruloyl)-beta-glucopyranoside (2), 3'-hydroxy, 6-O-methylscutellarein 7-O-beta-glucopyranoside (3), luteolin 7-O-beta-glucopyranoside (4), isoscutellarein 7-O-(6'-O-acetyl)-beta-allopyranosyl (1' --> 2')-beta-glucopyranoside (5), 3,4-dihydroxyphenethyl alcohol 8-O-beta-glucopyranoside (7), benzyl alcohol 7-O-beta-xylopyranosyl (1" --> 2')-beta-glucopyranoside (8), and (+)-syringaresinol 4'-O-beta-glucopyranoside (9). Compounds 2, 3 and 7-9 were reported for the first time in the genus Veronica. The structures of the isolates were determined by means of spectroscopic (UV, IR, 1D and 2D NMR, HR ESI-MS) methods. Isolated compounds (1-7) exhibited potent radical scavenging activity against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical.     

8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors, such as alpha7, alpha3beta4 and alpha4beta2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (100 nM), a linoleic acid derivative, potentiated responses of alpha7, alpha3beta4 and alpha4beta2 ACh receptors expressed in Xenopus oocytes that are blocked by 3-(1-[dimethylaminopropyl] indol-3-yl)-4-[indol-3-yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for alpha3beta4 ACh receptors. DCP-LA enhanced the nicotine-triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell-shaped dose-dependent manner at concentrations ranging from 10 nM to 10 microM, although DCP-LA by itself had no effect on GABA release. The DCP-LA action was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors, but not by mecamylamine or dihydro-beta-erithroidine, an inhibitor of alpha3beta4 and alpha4beta2 ACh receptors. A similar effect on GABA release was obtained with 12-O-tetradecanoylphorbol 13-acetate, a PKC activator. DCP-LA (100 nM) also enhanced GABA release triggered by choline, an agonist of alpha7 ACh receptors, but not 3-[2(s)-azetidinylmethoxy] pyridine, an agonist of alpha4beta2 ACh receptors. In addition, DCP-LA (100 nM) increased the rate of nicotine-triggered GABA(A) receptor-mediated miniature inhibitory post-synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or alpha-bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP-LA stimulates GABA release by enhancing activity of pre-synaptic alpha7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.     

Appropriate preoperative membranous urethral length predicts recovery of urinary continence after robot-assisted laparoscopic prostatectomy

Purpose

We investigated that preoperative membranous urethral length (MUL) would be associated with the recovery of urinary continence after robot-assisted laparoscopic prostatectomy (RALP).

Patients and methods

We studied 204 patients who underwent RALP between May 2013 and March 2016. All patients underwent pelvic magnetic resonance imaging (MRI) preoperatively to measure MUL. Urinary continence was defined as the use of one pad or less (safety pad). The 204 patients were divided into two groups: continence group, those who achieved recovery of continence at 3, 6, and 12?months after RALP, and incontinence group, those who did not. We retrospectively analyzed the patients in terms of preoperative clinical factors including age, body mass index (BMI), estimated prostate volume, neurovascular bundle salvage, history of preoperative hormonal therapy, and MUL.

Results

The safety pad use rate was 69.6%, 86.9%, and 91.1% at 3, 6, and 12?months, respectively. On univariate and multivariate analyses, MUL were significant factors in every term of recovery of urinary continence in both groups. According to the receiver operating characteristic (ROC) curve analysis, the preoperative MUL that could best predict early recovery of urinary continence at 3 months after RALP was 12?mm.

Conclusions

We suggest that preoperative MUL >?12?mm would be a predictor of early recovery of urinary continence after RALP.

     

The chemical NJ15 affects hypocotyl elongation and shoot gravitropism via cutin polymerization

We previously found a chemical, designated as NJ15, which inhibited both auxin and brassinosteroid responses in dark-grown Arabidopsis. To study its mode of action, we performed a phenotypic screening of NJ15-low-sensitive lines among mutant pools of Arabidopsis. One line (f127) showed clear NJ15-low-sensitivity in terms of hypocotyl elongation and shoot gravitropism. After further testing, it was determined that DCR, an enzyme involved in cutin polymerization, had lost its function in the mutant, which caused its low sensitivity to NJ15. Fatty acids are the base materials for polymers such as cutin and cuticular wax. We confirmed that NJ15 affects fatty acid biosynthesis, and that it does differently from cafenstrole, a known inhibitor of cuticular wax formation. Based on these results, we propose that the target of NJ15 is likely located within the cutin polymer formation pathway.

Abbreviations: Caf: cafenstrole; DEG: differentially expressed gene; FDR: false discovery rate; FOX: full length cDNA-overexpressor; VLCFA: very-long-chain fatty acid     

Synthesis and evaluation of a potent,well-balanced EP2/EP3 dual agonist

A highly potent and well-balanced dual agonist for the EP₂ and EP₃ receptors is described. Optimization of the lead compound was accomplished in consideration of the relative agonist activity against each EP subtype receptor and the pharmacokinetic profile. As the result, 2-[(2-{(1R,2R)-2-[(1E,4S)-5-cyclopentyl-4-hydroxy-4-methyl-1-penten-1-yl]-5-oxocyclopentyl}eth-yl)thio]-1,3-thiazole-4-carboxylic acid (10) showed excellent potency (human EC₅₀ EP₂?=?1.1?nM, EP₃?=?1.0?nM) with acceptable selectivity over the EP₁ and EP₄ subtypes (>2000-fold). Further fine-tuning of compound 10 led to identification of ONO-8055 as a clinical candidate. ONO-8055 was effective at an extremely low dose (0.01?mg/kg, po, bid) in rats, and dose-dependently improved voiding dysfunction in a monkey model of underactive bladder (UAB). ONO-8055 is expected to be a novel and highly promising drug for UAB.     

Sorting nexin 3, a protein upregulated by lithium, contains a novel phosphatidylinositol-binding sequence and mediates neurite outgrowth in N1E-115 cells

Lithium, a drug in the treatment of bipolar disorder, modulates many aspects of neuronal developmental processes such as neurogenesis, survival, and neuritogenesis. However, the underlying mechanism still remains to be understood. Here, we show that lithium upregulates the expression of sorting nexin 3 (SNX3), one of the Phox (PX) domain-containing proteins involved in endosomal sorting, and regulates neurite outgrowth in mouse N1E-115 neuroblastoma cells. The inhibition of SNX3 function by its knockdown decreases lithium-induced outgrowth of neurites. Transfection of the full-length SNX3 construct into cells facilitates the outgrowth. We also find that the C-terminus, as well as the PX domain, of SNX3 has a functional binding sequence with phosphatidylinositol monophosphates. Transfection of the C-terminal deletion mutant or only the C-terminus does not have an effect on the outgrowth. These results suggest that SNX3, a protein upregulated by lithium, is an as yet unknown regulator of neurite formation and that it contains another functional phosphatidylinositol phosphate-binding region at the C-terminus.     

Rab35, acting through ACAP2 switching off Arf6, negatively regulates oligodendrocyte differentiation and myelination

Oligodendrocyte precursor cells differentiate to produce myelin sheaths that insulate axons to ensure fast propagation of action potentials. Many aspects of differentiation are regulated by multiple extracellular signals. However, their intracellular signalings remain elusive. We show that Rab35 and its effector, ACAP2, a GTPase-activating protein that switches off Arf6 activity, negatively regulate oligodendrocyte morphological differentiation. Knockdown of Rab35 or ACAP2 with their respective small interfering RNAs promotes differentiation. As differentiation initiates, the activities of Rab35 and ACAP2 are down-regulated. The activity of Arf6, in contrast, is up-regulated. Arf6 knockdown inhibits differentiation, indicating that Rab35 and ACAP2 negatively regulate differentiation by down-regulating Arf6. Importantly, as differentiation proceeds, the activity of cytohesin-2, a guanine nucleotide exchange factor that switches on Arf6 activity, is up-regulated. Pharmacological inhibition of cytohesin-2 inhibits differentiation, suggesting that cytohesin-2 promotes differentiation by activating Arf6. Furthermore, using oligodendrocyte-neuronal cocultures, we find that knockdown of Rab35 or ACAP2 promotes myelination, whereas inhibition of cytohesin-2 or knockdown of Arf6 inhibits myelination. Thus Rab35/ACAP2 and cytohesin-2 antagonistically control oligodendrocyte differentiation and myelination through Arf6 regulation, presenting a unique small GTPase on/off switching mechanism.     

Activin A is an endogenous inhibitor of ureteric bud outgrowth from the Wolffian duct

Development of metanephric kidney begins with ureteric bud outgrowth from the Wolffian duct (WD). GDNF is believed to be a crucial positive signal in the budding process, but the negative regulation of this process remains unclear. Here, we examined the role of activin A, a member of TGF-beta family, in bud formation using an in vitro WD culture system. When cultured with the surrounding mesonephros, WDs formed many ectopic buds in response to GDNF. While the activin signaling pathway is normally active along the non-budding WD (as measured by expression of activin A and phospho-Smad2/3), activin A was absent and phospho-Smad2/3 was undetectable in the ectopic buds induced by GDNF. To examine the role of activin A in bud formation, we attempted to inactivate activin action. Interestingly, the addition of neutralizing anti-activin A antibody potentiated GDNF action. To further clarify the role of activin A, we also tested the effect of activin blockade on the WD cultured in the absence of mesonephros. WDs without mesonephros did not form ectopic buds even in the presence of GDNF. In contrast, blockade of activin action with a variety of agents acting through different mechanisms (natural antagonist, neutralizing antibodies, siRNA) enabled GDNF to induce ectopic buds. Inhibition of GDNF-induced bud formation by activin A was accompanied by inhibition of cell proliferation, reduced expression of Pax-2, and decreased phosphorylation of PI3-kinase and MAP kinase in the WD. Our data suggest that activin A is an endogenous inhibitor of bud formation and that cancellation of activin A autocrine action may be critical for the initiation of this process.