nM-1 microM Research Articles

Effects of coating materials on two dimensional stress-induced deflection of nanomechanical sensors.

Nanomechanical sensors, which have been expected as a promising platform for various applications, will not generate any measureable signal without an appropriate coating (receptor) layer. In spite of this critical dependence on the coating layer, there is almost no guideline for the optimization of these layers in terms of their material properties; such as, Young's modulus, Poisson's ratio, and geometrical parameters. In the present study, the effects of coating layer thickness [3 nm-10 microm], Young's modulus [100 Pa to approximately 1 TPa], and Poisson's ratio [0.10 to approximately 0.45] are investigated with the finite element analysis (FEA), focusing on systems with two dimensional (2D) stress induced either on top of the coating layer (i.e., surface stress) or at the interface between the coating layer and the silicon cantilever. It is found that the coating layer can either enhance or reduce the deflection of nanomechanical sensors depending on its material properties. These results provide guidelines for designing a coating layer to achieve higher sensitivity.

Salmeterol inhibits anaphylactic histamine release from guinea-pig isolated mast cells.

Salmeterol (1 nM-100 microM) showed an inhibitory action on anaphylactic histamine release from mast cells, isolated from pleural and peritoneal cavities of actively sensitized guinea-pigs and stimulated by incubation with allergen. The effect is concentration-dependent and is reduced by the beta-adrenoceptor antagonist propranolol (1 microM). This study supports the hypothesis of an anti-inflammatory property of salmeterol, which concerns cells involved in the early phases of asthma.

Modulation of (-)-sulpiride-induced increase in electrically-evoked release of dopamine from rat striatal slices.

(-)-Sulpiride (10 nM-10 microM) in the superfusate, dose-dependently increased the electrically-evoked release of dopamine from rat striatal slices. (+)-Sulpiride had little effect on evoked release of dopamine up to 10 microM. Apomorphine inhibited electrically evoked release of dopamine, and this effect of apomorphine was antagonized by (-)-sulpiride. SCH23390 and forskolin had no effect on the (-)-sulpiride-induced increase in evoked release of dopamine. Treatment with the irreversible dopamine-receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline caused a significant increase in evoked release of dopamine and prevented the (-)-sulpiride-induced increase in the evoked release of dopamine. These results indicate that the (-)-sulpiride-induced increase in evoked release of dopamine is due to antagonism of the activation of dopamine autoreceptors by endogenously released dopamine.

Initial testing (stage 1) of AZD6244 (ARRY‐142886) by the pediatric preclinical testing program

AZD6244 (ARRY-142886) is a potent small molecule inhibitor of MEK1/2 that is in phase 2 clinical development. AZD6244 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro panel (1 nM-10 microM). In vivo AZD6244 was tested at a dose of 100 mg/kg administered orally twice daily 5 days per week for 6 weeks. Subsequently, AZD6244 was evaluated against two juvenile pilocytic astrocytoma (JPA) xenografts using once and twice daily dosing schedules. Phosphorylation of ERK1/2 was used as a surrogate for in vivo inhibition of MEK1/2 was determined by immunoblotting. At the highest concentration used in vitro (10 microM) AZD6244 only inhibited growth by 50% in 5 of the 23 cell lines. Against the in vivo tumor panels, AZD6244 induced significant differences in EFS distribution in 10 of 37 (27%) solid tumor models and 0 of 6 acute lymphoblastic leukemia (ALL) models. There were no objective responses. Pharmacodynamic studies indicated at this dose and schedule AZD6244 completely inhibited ERK1/2 phosphorylation. AZD6244 was evaluated against two JPA xenografts, BT-35 (wild-type BRAF) and BT-40 (mutant [V600E] BRAF). BT-40 xenografts were highly sensitive to AZD6244, whereas BT-35 xenografts progressed on AZD6244 treatment. At the dose and schedule of administration used, AZD6244 as a single agent had limited in vitro and in vivo activity against the PPTP tumor panels despite inhibition of MEK1/2 activity. However, AZD6244 was highly active against BT-40 JPA xenografts that harbor constitutively activated BRAF, causing complete regressions.

2‐arachidonyl glycerol activates platelets via conversion to arachidonic acid and not by direct activation of cannabinoid receptors

There are conflicting views in the literature as to whether cannabinoids have an impact on platelet activity and to what extent cannabinoid receptors are involved. This is an important issue to resolve because platelet effects of putative therapeutic cannabinoid inhibitors and stimulators will have an impact on their potential benefits and safety. The data presented in this manuscript clearly show that the endocannabinoid 2-arrachidonyl glycerol can activate platelet activity, but that the effects are mediated through an aspirin-sensitive pathway that is not affected by cannabinoid receptor antagonists or FAAH inhibition, but is abolished by MAGL inhibition. The findings question the role of cannabinoid receptors in platelet function and suggest that platelet function is unlikely to be directly affected by cannabinoid receptor antagonists, at least in the acute phase. Cannabinoid receptor-1 (CB(1)) antagonists suppress appetite and induce weight loss. Direct antagonism of CB(1) receptors on platelets might be an additional benefit for CB(1) antagonists, but the role of CB(1) receptors in platelets is controversial. We tested the hypothesis that the endocannabinoid, 2-arachidonyl glycerol (2-AG), induces platelet aggregation by a COX-mediated mechanism rather than through CB(1) receptor activation, in blood obtained from healthy volunteers and patients with coronary artery disease receiving low dose aspirin. Aggregatory responses to the cannabinoids 2-AG and Delta(9)-THC were examined in blood sampled from healthy volunteers (n= 8) and patients (n= 12) with coronary artery disease receiving aspirin using whole blood aggregometry. The effects of CB(1) (AM251) and CB(2) (AM630) antagonists, as well as fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL) inhibitors and aspirin on 2-AG-induced aggregation were also assessed. AM251 (100 nm-30 microm) had no effect on platelet aggregation induced by either ADP (P= 0.90) or thrombin (P= 0.86). 2-AG, but not Delta(9)-THC, induced aggregation. 2-AG-induced aggregation was unaffected by AM251 and AM630 but was abolished by aspirin (P < 0.001) and by the MAGL inhibitor, URB602 (P < 0.001). Moreover, the aggregatory response to 2-AG was depressed (by >75%, P < 0.001) in blood from patients with coronary artery disease receiving aspirin compared with that from healthy volunteers. 2-AG-mediated activation of platelets is via metabolism to arachidonic acid by MAGL, and not through direct action on CB(1) or CB(2) receptors, at least in the acute phase.

Signalling pathways involved in sildenafil‐induced relaxation of human bladder dome smooth muscle

The mechanism(s) of action responsible for the beneficial effects of phosphodiesterase 5 (PDE5) inhibitors including sildenafil on lower urinary tract symptoms suggestive of benign prostate hyperplasia are unclear. In particular, the role of the NO-cGMP signalling pathway in regulating human bladder dome smooth muscle relaxation is questionable. Thus, we assessed the ability of a PDE5 inhibitor, sildenafil, to relax such tissue, and identified the signalling pathways involved in this relaxation. Human bladder samples were obtained from 20 patients with no overactive bladder undergoing cystectomy for bladder cancer. Detrusor strips were mounted isometrically in Krebs-HEPES solution. Concentration-response curves for sildenafil (10 nM-30 microM) were generated in the presence of various inhibitors on carbachol-induced pre-contraction. Sildenafil relaxed carbachol-pre-contracted human detrusor strips, starting at 3 microM. This effect was not modified by NO donors, S-nitroso-N-acetylpenicillamine (10 microM) or sodium nitroprusside (300 nM), but was significantly inhibited by inhibition of guanylate cyclase (with ODQ, 10 microM) or adenylyl cyclase (with MDL-12,330A, 10 microM), by the ATP-sensitive potassium channel inhibitor, glibenclamide (10 microM), or inhibition of the large (with iberiotoxin, 30 nM) or small (with apamin, 100 nM) conductance calcium-activated potassium channels. Sildenafil-induced relaxation of human detrusor smooth muscle involved cGMP-, cAMP- and K(+) channel-dependent signalling pathways, with a minor contribution from NO. The effect of this sildenafil-induced relaxation on the clinical benefit of PDE5 inhibitors on urinary storage symptoms in men deserves further investigation.

Urotensin II: A Novel Target in Human Corpus Cavernosum

Urotensin II (U-II) is a cyclic peptide originally isolated from the teleost neurosecretory system and subsequently identified in other species, including man. U-II was identified as the natural ligand of an orphan G-protein coupled receptor (UT receptor). U-II and UT receptor are expressed in a variety of peripheral organs and especially in cardiovascular tissue. U-II caused both constrictor and vasodilator effect, depending by vascular bed. The in vivo functional consequences of U-II on the cardiovascular hemodynamics are not clearly understood. To investigate the presence of UT receptor and the effect of U-II in human corpus cavernosum (HCC) strips. To evaluate the effect of U-II in vivo in anesthetized rats. UT receptor expression as protein and as mRNA were assessed by Western blot and reverse transcriptase polymerase chain reaction. Next, the UT receptor localization was evaluated by immunohistochemical analysis. By using HCC strips, with or without endothelium, the effect of U-II (0.1 nM-10 microM) was evaluated. In order to asses the nitric oxide (NO) involvement, the strips were incubated with N (G)-nitro-L-arginine methyl ester (NO synthase inhibitor, 100 microM). U-II (0.1, 0.3, 1.0 nmol/rat) effect in vivo was studied in anesthetized rats by monitoring the intracavernous and systemic blood pressure. HCC expresses the UT receptor and its activation, by UII, causes an endothelium- and NO-dependent relaxation. UT receptor is expressed in human and rat corpus cavernosum. In HCC UT receptor is localized on endothelial cells. U-II significantly relaxed HCC strips in endothelium- and -NO-dependent fashion. The peptide caused a significant increase in intracavernous pressure in anesthetized rats. This study demonstrates that UT receptor is expressed on the endothelium of HCC. U-II/UT receptor system is involved in HCC function and it involves endothelium and NO pathway. Thus, U-II/UT receptor pathway could be involved in erectile function.

Stimulatory effects of the soy phytoestrogen genistein on noradrenaline transporter and serotonin transporter activity

We examined the effects of genistein, one of the major soy phytoestrogens, on the activity of noradrenaline transporter (NAT) and serotonin transporter. Treatment with genistein (10 nM-10 microM) for 20 min stimulated [(3)H]noradrenaline (NA) uptake by SK-N-SH cells. Genistein also stimulated [(3)H]NA uptake and [(3)H]serotonin uptake by NAT and serotonin transporter transiently transfected COS-7 cells, respectively. Kinetics analysis of the effect of genistein on NAT activity in NAT-transfected COS-7 cells revealed that genistein significantly increased the maximal velocity of NA transport with little or no change in the affinity. Scatchard analysis of [(3)H]nisoxetine binding to NAT-transfected COS-7 cells showed that genistein increased the maximal binding without altering the dissociation constant. Although genistein is also known to be an inhibitor of tyrosine kinases, daidzein, another soy phytoestrogen and an inactive genistein analogue against tyrosine kinases, had little effect on [(3)H]NA uptake by SK-N-SH cells. The stimulatory effects on NAT activity were observed by treatment of tyrphostin 25, an inhibitor of epidermal growth factor receptor tyrosine kinase, whereas orthovanadate, a protein tyrosine phosphatase inhibitor, suppressed [(3)H]NA uptake by NAT-transfected COS-7 cells. These findings suggest that genistein up-regulates the activity of neuronal monoamine transporters probably through processes involving protein tyrosine phosphorylation.

Uric Acid Does Not Affect the Acetylcholine-Induced Relaxation of Aorta from Normotensive and Deoxycorticosterone Acetate-Salt Hypertensive Rats

Uric acid (UA) results from xanthine oxidase (XO) catabolism of xanthine and is the final product of purine catabolism in humans. In this species, hyperuricemia is associated with gout, nephropathy, and increased cardiovascular disease risk. Although the effects of hyperuricemia in vascular biology are overall controversial, UA has been described as an antioxidant and as potentially improving endothelial function. Hypertension is associated with endothelial dysfunction. We hypothesized that UA improves the endothelial function of aorta from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. UA (100 microM) in the presence of the uricase inhibitor oxonic acid (10 microM) did not modify relaxation to acetylcholine (ACh) (1 nM-10 microM) in the aorta from nontreated, sham normotensive, and DOCA-salt hypertensive rats [response to 10 microM ACh for UA versus vehicle, respectively: nontreated = 37 +/- 7 versus 48 +/- 7%, sham = 53 +/- 15 versus 57 +/- 20%, DOCA = 81 +/- 4 versus 85 +/- 2% from 20 microM prostaglandin 2alpha (PGF(2alpha))-induced contraction]. Allopurinol (100 microM), a XO inhibitor, did not significantly alter the ACh-induced relaxation of sham and DOCA aortic rings (response to 10 microM ACh for allopurinol versus vehicle, respectively: sham = 61 +/- 5 versus 68 +/- 9%, DOCA = 87 +/- 6 versus 88 +/- 3% from 20 microM PGF(2alpha)-induced contraction). Uricemia, ranging from unmeasurable to 547 microM in sham and to 506 microM in DOCA rats, was not significantly different between these two groups. The expression and activity of XO, as well as the expression of uricase, were not different between sham and DOCA rat aorta. We conclude that, at least in vitro, UA does not affect the ACh-induced relaxation of normotensive and DOCA-salt hypertensive rats.

Characterisation of the prostanoid receptor mediating inhibition of smooth muscle contractility in the rat prostate gland

This study characterised the inhibitory actions of prostaglandins on smooth muscle contractility in the rat prostate gland. Immunohistochemical studies were carried out to identify and localise the two isoforms of cyclooxygenase (COX) enzyme and the subtypes of prostanoid receptors present in the rat prostate. Isolated organ bath studies were carried out to pharmacologically characterise the subtype of prostanoid receptor mediating the inhibitory effects of prostanoids on the rat prostate. Immunohistochemical studies confirmed the presence of mainly COX-2 within the prostatic stroma. Isolated organ bath studies showed that prostaglandin E(2) (PGE(2); 10 nM-10 microM) but not prostaglandin D(2) (10 nM-10 microM), PGF2alpha (10 nM-10 microM), prostacyclin (10 nM-10 microM) or U46619 (10 nM-10 microM) inhibited nerve-mediated contractile responses to electrical field stimulation. Similarly, sulprostone (10 nM-10 microM) had no affect on the magnitude of the electrically evoked contractions. PGE(2) (0.1-10 microM) did not affect contractions elicited by noradrenaline or adenosine 5'-triphosphate. PGE(2)-mediated inhibition of electrical field stimulation induced contractions was attenuated by AH 6809 (10 microM) but not SC 19220 (10 microM) or AH 23848 (10 microM). It is concluded that prostaglandins can inhibit contractions of the rat prostate gland through a prostanoid receptor of the EP(2) subtype.

Inhibition of ATP-Induced Ca2+ Influx by Corticosterone in Dorsal Root Ganglion Neurons

In addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects on neurons. In the present study, the effect of corticosterone (CORT) on ATP-induced Ca(2+) mobilization in cultured dorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca(2+)]i). ATP, an algesic agent, caused [Ca(2+)]i increase in DRG neurons by activation of P2X receptor. Pretreatment with CORT (1 nM-1 microM for 5 min) inhibited ATP-induced [Ca(2+)]i increase in DRG neurons. The rapid inhibition of ATP-induced Ca(2+) response by CORT was concentration-dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486 (10 microM). Furthermore, the inhibitory effect of CORT was abolished by protein kinase A inhibitor H89 (10 microM), but was not influenced by protein kinase C inhibitor Chelerythrine chloride (10 microM). On the other hand, membrane-impermeable bovine serum albumin-conjugated corticosterone had no effect on ATP-induced [Ca(2+)]i transients. These observations suggest that a nongenomic pathways may be involved in the effect of CORT on ATP-induced [Ca(2+)]i transients in cultured DRG neurons.

High Functional Expression of Osteoblasts on Imogolite, Aluminosilicate Nanotubes

Imogolite is a naturally occurring aluminosilicate clay mineral with a nanotube structure of 2 nm diameter and 60 nm-2 microm length. In this study, synthetic imogolite nanotubes were applied to cell culture and the properties as a scaffold were compared with those of a conventional culture dish and a carbon nanotube (CNT) scaffold. The surface characteristics of the imogolite scaffold were drastically changed with the amount of coated imogolite on the dish. With the increase of imogolite concentration, the surface morphology of the scaffolds changed from an island-like shape in random orientation to a self-organized fiber texture aligned in the same direction, and finally full coverage in a random orientation with plural layers. Silicon releasability of the scaffolds was increased with the amount of coated imogolite. Mouse osteoblast-like cells (MC3T3-E1) cultured on imogolite showed a flat-form morphology and a cytoskeleton developed in all directions in contrast to a spindle-shaped morphology developed in one direction on cell culture dishes and CNT scaffolds. The proliferation and osteoblastic functions in ALP activity and mineralization showed similar increasing tendency. The largest values were obtained from the scaffold with a high concentration of imogolite. The results indicated good biocompatibility between the cells and imogolite scaffold and the enhancement effect of proliferation and osteoblastic differentiation.

Oxytocin receptors in the primate ovary: molecular identity and link to apoptosis in human granulosa cells.

Oxytocin (OT) is produced by granulosa cells (GCs) of pre-ovulatory ovarian follicles and the corpus luteum (CL) in some mammalian species. Actions of OT in the ovary have been linked to luteinization, steroidogenesis and luteolysis. Human IVF-derived (h)GCs possess a functional OT receptor (OTR), linked to elevation of intracellular Ca(2+), but molecular identity of the receptor for OT in human granulosa cells (hGCs) and down-stream consequences are not known. RT-PCR, sequencing and immunocytochemistry identified the genuine OTR in hGCs. OT (10 nM-10 microM) induced elevations of intracellular Ca(2+) levels (Fluo-4 measurements), which were blocked by tocinoic acid (TA; 50 microM, a selective OTR-antagonist). Down-stream effects of OTR-activation include a concentration dependent decrease in cell viability/metabolism, manifested by reduced ATP-levels, increased caspase3/7-activity (P < 0.05) and electron microscopical signs of cellular regression. TA blocked all of these changes. Immunoreactive OTR was found in the CL and GCs of large and, surprisingly, also small pre-antral follicles of the human ovary. Immunoreactive OTR in the rhesus monkey ovary was detected in primordial and growing primary follicles in the infantile ovary and in follicles at all stages of development in the adult ovary, as well as the CL: these results were corroborated by RT-PCR analysis of GCs excised by laser capture microdissection. Our study identifies genuine OTRs in human and rhesus monkey GCs. Activation by high levels of OT leads to cellular regression in hGCs. As GCs of small follicles also express OTRs, OT may have as yet unknown functions in follicular development.

Involvement of P2X7Receptors in the Hypoxia-Induced Death of Rat Retinal Neurons

To investigate the hypoxia-induced death of rat retinal neurons and to determine whether P2X(7) activation is involved in this type of neuronal death. Cultured retinal neurons from fetal rats were used. The effects and time course of various degrees of hypoxia (1%-5% O(2)) in the death of retinal neurons, were examined. The effects of P2X(7) antagonists, oxidized adenosine triphosphate (oxidized ATP; 30-100 microM), and brilliant blue G (BBG; 100 nM-10 microM) on hypoxia-induced neuronal death, including apoptosis, were assessed by using trypan blue exclusion, TUNEL assays, and cleaved caspase-3 immunoreactivity. Immunocytochemical analysis was performed to determine whether these neurons express P2X(7) receptors. The effects of P2X(7) receptor stimulation, induced by the P2X(7) agonist benzoyl- benzoyl-ATP (BzATP), on neuronal viability and intracellular Ca(2+) levels ([Ca(2+)](i)) were examined. Retinal neuronal death increased according to the degree of hypoxia and became more severe after 12 hours. Both oxidized ATP and BBG significantly decreased hypoxia-induced neuronal death. Immunocytochemistry demonstrated that P2X(7) receptors were expressed by the cultured retinal neurons. ATP and BzATP caused P2X(7) receptor-dependent neuronal death in a dose-dependent manner and led to a sustained increase in [Ca(2+)](i), with BzATP being more effective than ATP. These effects were hypoxia-induced factor-1alpha- independent and were prevented by oxidized ATP. The results suggest that the death of retinal neurons can be triggered by hypoxia and that P2X(7) activation is involved in the hypoxia-induced death of retinal neurons. P2X(7) antagonists can prevent hypoxia-induced damage in retinal neurons.

Mechanisms involved in the regulation of bovine pulmonary vascular tone by the 5‐HT1B receptor

5-HT(1B) receptors may have a role in pulmonary hypertension. Their relationship with the activity of BK(Ca,) a T-type voltage-operated calcium channel (VOCC) and cyclic nucleotide-mediated relaxation was examined. Ring segments of bovine pulmonary arteries were mounted in organ baths in modified Krebs-Henseleit buffer (37 degrees C) under a tension of 20 mN and gassed with 95% O(2)/5% CO(2). Isometric recordings were made using Chart 5 software. Contractile responses to 5-HT (10 nM-300 microM) were inhibited similarly by the 5-HT(1B) receptor antagonist SB216641 (100 nM) and the T-type VOCC blockers mibefradil (10 microM) and NNC550396 (10 microM) with no additive effect between SB216641 and mibefradil. Inhibition by SB216641 was prevented by the potassium channel blocker, charybdotoxin (100 nM). 5-HT(1B) receptor activation and charybdotoxin produced a mibefradil-sensitive potentiation of responses to U46619. Bradykinin (0.1 nM-30 microM), sodium nitroprusside (0.01 nM-3 microM), zaprinast (1 nM-3 microM), isoprenaline (0.1 nM-10 microM) and rolipram (1 nM-3 microM) produced 50% relaxation of arteries constricted with 5-HT (1-3 microM) or U46619 (30-50 nM) in the presence of 5-HT(1B) receptor activation, but full relaxation of arteries constricted with U46619, the 5-HT(2A) receptor agonist 2,5 dimethoxy-4 iodoamphetamine (1 microM) or 5-HT in the presence of 5-HT(1B) receptor antagonism. Enhanced relaxation of 5-HT-constricted arteries by cGMP-dependent pathways, seen in the presence of the 5-HT(1B) receptor antagonist, was reversed by charybdotoxin whereas cAMP-dependent relaxation was only partly reversed by charybdotoxin. 5-HT(1B) receptors couple to inhibition of BK(Ca), thus increasing tissue sensitivity to contractile agonists by activating a T-type VOCC and impairing cGMP-mediated relaxation. Impaired cAMP-mediated relaxation was only partly mediated by inhibition of BK(Ca).

Increased colonic motility in a rat model of irritable bowel syndrome is associated with up-regulation of L-type calcium channels in colonic smooth muscle cells

This paper aimed to investigate the relationship between up-regulation of L-type calcium channels and altered motility disorder in a rat model of irritable bowel syndrome (IBS). Male Sprague-Dawley rats were subjected to neonatal maternal separation (NMS) from postnatal day 2-14 or normal handling (NH), and used when weighted 250-300 g. Colonic smooth muscle contractions was studied in an organ bath system. L-type Ca(2+) channel alpha(1c) subunit expression in smooth muscles from rat colon were studied by immunofluorescence and Western blotting analysis. The intracellular calcium concentration ([Ca(2+)](i)) of enzymatically isolated single colonic smooth muscle cell was studied with laser confocal fluorescent microscopy. The fecal pellets during 1 h water avoidance stress (WAS) were significantly increased; the amplitude of spontaneous contractions and contractions induced by Bay K 8644 (10 nM-1 microM), KCl (10-60 mM) and ACh (100 nM-10 microM) were significantly increased in NMS rats, when comparing with that of NH rats. [Ca(2+)]i induced by Bay K 8644 (1 microM), KCl (40 mM), and ACh (10 microM) significantly increased in muscle cells of NMS rats than NH rats. Further, alpha(1c) protein expression was significantly up-regulated in colonic smooth muscle of NMS rats than NH rats. These results suggest that NMS lead to up-regulation of L-type Ca(2+) channels expression in the colon, which contributes to the colonic motility disorder. Our findings provide direct evidence to help understanding the underlying mechanism of chronic stress-induced colonic motility disorder in IBS.

Superhydrophobic surfaces by hybrid raspberry-like particles

Surface roughness on different length scales is favourable for superhydrophobic behaviour of surfaces. Here we report (i) an improved synthesis for hybrid raspberry-like particles and (ii) a novel method to obtain superhydrophobic films of good mechanical stability. Polystyrene spheres with a diameter of 400 nm-1 microm are decorated with silica colloids < 100 nm in size, thus introducing surface asperities on a second length scale. To improve mechanical resistance, we then coated the polystyrene core and attached silica colloids with a smooth silica shell of 10 nm to 40 nm thickness. All three steps of this synthesis procedure can be sensitively tuned so that the average size and number of the silica colloids as well as the morphology of the resulting raspberry particles can be predicted. As the particles disperse in water, either monolayers can be prepared by dip coating or multilayers by drop casting. Although mechanically stable, the shells are porous enough to allow for leakage of molten or dissolved polystyrene from the core. In tetrahydrofuran vapour polystyrene bridges form between the particles that render the multilayer-film stable. Leaked polystyrene that masks some asperities can be removed by plasma cleaning. Surface roughness on larger scales can be tuned by the drying procedure. The films are hydrophobized by silanization with a semi-fluorinate silane.

New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in 3D cancer spheroids induced by purified colon tumor-initiating cells

BackgroundGrowing evidence suggests that the majority of tumors are organized hierarchically, comprising a population of tumor-initiating, or cancer stem cells (CSCs) responsible for tumor development, maintenance and resistance to drugs. Previously we have shown that the CD133high/CD44high fraction of colon cancer cells is different from their bulk counterparts at the functional, morphological and genomic levels. In contrast to the majority of colon cancer cells expressing moderate levels of CD133, CD44 and CD166, cells with a high combined expression of CD133 and CD44 possessed several characteristic stem cell features, including profound self-renewal capacity in vivo and in vitro, and the ability to give rise to different cell phenotypes. The present study was undertaken for two aims: a) to determine stem cell-related genomic characteristics of floating 3D multicellular spheroids induced by CD133high/CD44high colon cancer cells; and b) to evaluate CSC-specific alterations induced by new-generation taxoid SB-T-1214.ResultsSelected CSC phenotype was isolated from three independent invasive colon cancer cell lines, HCT116, HT29 and DLD-1. A stem cell-specific PCR array assay (SABiosciences) revealed that colonospheres induced by purified CD133high/CD44high expressing cells display profound up-regulation of stem cell-related genes in comparison with their bulk counterparts. The FACS analysis has shown that the 3D colonospheres contained some minority cell populations with high levels of expression of Oct4, Sox2, Nanog and c-Myc, which are essential for stem cell pluripotency and self-renewal. Single administration of the SB-T-1214 at concentration 100 nM-1 μM for 48 hr not only induced growth inhibition and apoptotic cell death in these three types of colon cancer spheroids in 3D culture, but also mediated massive inhibition of the stem cell-related genes and significant down-regulation of the pluripotency gene expression. PCR array and FACS data were confirmed with western blotting. Importantly, viable cells that survived this treatment regimen were no longer able to induce secondary floating spheroids and exhibited significant morphological abnormalities.ConclusionsWe report here that a new-generation taxoid SB-T-1214 possesses significant activity against colon cancer spheroids induced by and enriched with drug resistant tumorigenic CD133high/CD44high cells and efficiently inhibited expression of the majority of stem cell-related genes. Our data indicates that the previously observed long-term efficacy of SB-T-1214 against drug resistant colon tumors in vivo may be explained by the down-regulation of multiple stem cell-related genes in the tumorigenic cell population, in addition to its known efficacy as a mitotic poison against proliferating cancer cells.

Chemotactic and Cytotoxic Effects of Minocycline on Human Retinal Pigment Epithelial Cells

To reveal the effects of minocycline, an anti-inflammatory and neuroprotective agent, on the viability and physiological properties of retinal pigment epithelial (RPE) cells and to compare the effects with those of triamcinolone acetonide. The proliferation of human RPE cells in vitro was investigated with a bromodeoxyuridine immunoassay; chemotaxis was examined with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. The gene expression of growth factors and MMP-9 was determined with real-time RT-PCR, and the secretion of VEGF was examined with ELISA. The phosphorylation of p38 MAPK and ERK1/2 proteins was determined with Western blot analysis. Minocycline at low concentrations (50 nM-20 microM) stimulated chemotaxis and decreased the proliferation of RPE cells. Minocycline at high concentrations (above 5 microM) decreased the viability of RPE cells through the induction of cell necrosis. The chemotactic effect of minocycline was mediated by the stimulation of autocrine PDGF signaling and the activation of p38 MAPK. Minocycline promoted the expression of PDGF-B, HGF, VEGF, and MMP-9 and increased the amounts of phosphorylated p38 and ERK1/2 proteins in RPE cells. Triamcinolone reduced PDGF-evoked chemotaxis and VEGF expression and secretion and had no significant effects on cell viability and proliferation. Triamcinolone did not reverse the effects of minocycline on cell proliferation, chemotaxis, or viability or the expression of VEGF. Low-dose minocycline induces the activation of RPE cells, as indicated by the activation of p38 and ERK1/2 and by enhanced chemotaxis mediated by autocrine PDGF signaling. High-dose minocycline induces RPE cell degeneration.

Orexins Excite Neurons of the Rat Cerebellar Nucleus Interpositus Via Orexin 2 Receptors In Vitro

Orexins are newfound hypothalamic neuropeptides implicated in the regulation of feeding behavior, sleep-wakefulness cycle, nociception, addiction, emotions, as well as narcolepsy. However, little is known about roles of orexins in motor control. Therefore, the present study was designed to investigate the effect of orexins on neuronal activity in the cerebellum, an important subcortical center for motor control. In this study, perfusing slices with orexin A (100 nM-1 microM) or orexin B (100 nM-1 microM) both produced neurons in the rat cerebellar interpositus nucleus (IN) a concentration-dependent excitatory response (96/143, 67.1%). Furthermore, both of the excitations induced by orexin A and B were not blocked by the low-Ca(2+)/high-Mg(2+) medium (n = 8), supporting a direct postsynaptic action of the peptides. Highly selective orexin 1 receptor antagonist SB-334867 did not block the excitatory response of cerebellar IN neurons to orexins (n = 22), but [Ala(11), D-Leu(15)] orexin B, a highly selective orexin 2 receptor (OX(2)R) agonist, mimicked the excitatory effect of orexins on the cerebellar neurons (n = 18). These results demonstrate that orexins excite the cerebellar IN neurons through OX(2)R and suggest that the central orexinergic nervous system may actively participate in motor control through its modulation on one of the final outputs of the spinocerebellum.