A highly efficient sensor platform using simply manufactured nanodot patterned substrates

Block copolymer (BCP) self-assembly is a low-cost means to nanopattern surfaces. Here, we use these nanopatterns to directly print arrays of nanodots onto a conducting substrate (Indium Tin Oxide (ITO) coated glass) for application as an electrochemical sensor for ethanol (EtOH) and hydrogen peroxide (H

The neuropeptide pituitary adenylate cyclase activating protein stimulates human monocytes by transactivation of the Trk/NGF pathway.

Transactivation is a process whereby stimulation of G-protein-coupled receptors (GPCR) activates signaling from receptors tyrosine kinase (RTK). In neuronal cells, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) acting through the GPCR VPAC-1 exerts trophic effects by transactivating the RTK TrkA receptor for the nerve growth factor (NGF). Both PACAP and NGF have pro-inflammatory activities on monocytes. We have tested the possibility that in monocytes, PACAP, as reported in neuronal cells, uses NGF/TrkA signaling pathway. In these cells, PACAP increases TrkA tyrosine phosphorylations through a PI-3kinase dependent but phospholipase C independent pathway. K252a, an inhibitor of TrkA decreases PACAP-induced Akt and ERK phosphorylation and calcium mobilisation resulting in decreases in intracellular H2O2 production and membrane upregulation of CD11b expression, both functions being inhibited after anti-NGF or anti-TrkA antibody treatment. K252a also inhibits PACAP-associated NF-KB activity. Monocytes increase in NGF production is seen after micromolar PACAP exposure while nanomolar treatment which desensitizes cells to high dose of PACAP prevents PACAP-induced TrkA phosphorylation, H2O2 production and CD11b expression. Finally, NGF-dependent ERK activation and H2O2 production is pertussis toxin sensitive. Altogether these data indicate that in PACAP-activated monocytes some pro-inflammatory activities occur through transactivation mechanisms involving VPAC-1, NGF and TrkA-associated tyrosine kinase activity.

Anti-cholinergic load, health care utilization, and survival in people with advanced cancer: a pilot study.

INTRODUCTION: Anti-cholinergic medications have been associated with increased risks of cognitive impairment, premature mortality and increased risk of hospitalisation. Anti-cholinergic load associated with medication increases as death approaches in those with advanced cancer, yet little is known about associated adverse outcomes in this setting. METHODS: A substudy of 112 participants in a randomised control trial who had cancer and an Australia modified Karnofsky Performance Scale (AKPS) score (AKPS) of 60 or above, explored survival and health service utilisation; with anti-cholinergic load calculated using the Clinician Rated Anti-cholinergic Scale (modified version) longitudinally to death. A standardised starting point for prospectively calculating survival was an AKPS of 60 or above. RESULTS: Baseline entry to the sub-study was a mean 62 +/- 81 days (median 37, range 1-588) days before death (survival), with mean of 4.8 (median 3, SD 4.18, range 1 - 24) study assessments in this time period. Participants spent 22% of time as an inpatient. There was no significant association between anti-cholinergic score and time spent as an inpatient (adjusted for survival time) (p = 0.94); or survival time. DISCUSSION: No association between anti-cholinergic load and survival or time spent as an inpatient was seen. Future studies need to include cognitively impaired populations where the risks of symptomatic deterioration may be more substantial.

Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist.

We developed a high-throughput yeast-based assay to screen for chemical inhibitors of Ca(2+)/calmodulin-dependent kinase pathways. After screening two small libraries, we identified the novel antagonist 125-C9, a substituted ethyleneamine. In vitro kinase assays confirmed that 125-C9 inhibited several calmodulin-dependent kinases (CaMKs) competitively with Ca(2+)/calmodulin (Ca(2+)/CaM). This suggested that 125-C9 acted as an antagonist for Ca(2+)/CaM rather than for CaMKs. We confirmed this hypothesis by showing that 125-C9 binds directly to Ca(2+)/CaM using isothermal titration calorimetry. We further characterized binding of 125-C9 to Ca(2+)/CaM and compared its properties with those of two well-studied CaM antagonists: trifluoperazine (TFP) and W-13. Isothermal titration calorimetry revealed that binding of 125-C9 to CaM is absolutely Ca(2+)-dependent, likely occurs with a stoichiometry of five 125-C9 molecules to one CaM molecule, and involves an exchange of two protons at pH 7.0. Binding of 125-C9 is driven overall by entropy and appears to be competitive with TFP and W-13, which is consistent with occupation of similar binding sites. To test the effects of 125-C9 in living cells, we evaluated mitogen-stimulated re-entry of quiescent cells into proliferation and found similar, although slightly better, levels of inhibition by 125-C9 than by TFP and W-13. Our results not only define a novel Ca(2+)/CaM inhibitor but also reveal that chemically unique CaM antagonists can bind CaM by distinct mechanisms but similarly inhibit cellular actions of CaM.

BMP signaling in the development of the mouse esophagus and forestomach.

The stratification and differentiation of the epidermis are known to involve the precise control of multiple signaling pathways. By contrast, little is known about the development of the mouse esophagus and forestomach, which are composed of a stratified squamous epithelium. Based on prior work in the skin, we hypothesized that bone morphogenetic protein (BMP) signaling is a central player. To test this hypothesis, we first used a BMP reporter mouse line harboring a BRE-lacZ allele, along with in situ hybridization to localize transcripts for BMP signaling components, including various antagonists. We then exploited a Shh-Cre allele that drives recombination in the embryonic foregut epithelium to generate gain- or loss-of-function models for the Bmpr1a (Alk3) receptor. In gain-of-function (Shh-Cre;Rosa26(CAG-loxpstoploxp-caBmprIa)) embryos, high levels of ectopic BMP signaling stall the transition from simple columnar to multilayered undifferentiated epithelium in the esophagus and forestomach. In loss-of-function experiments, conditional deletion of the BMP receptor in Shh-Cre;Bmpr1a(flox/flox) embryos allows the formation of a multilayered squamous epithelium but this fails to differentiate, as shown by the absence of expression of the suprabasal markers loricrin and involucrin. Together, these findings suggest multiple roles for BMP signaling in the developing esophagus and forestomach.

A longitudinal study of epigenetic variation in twins.

DNA methylation is a key epigenetic mechanism involved in the developmental regulation of gene expression. Alterations in DNA methylation are established contributors to inter-individual phenotypic variation and have been associated with disease susceptibility. The degree to which changes in loci-specific DNA methylation are under the influence of heritable and environmental factors is largely unknown. In this study, we quantitatively measured DNA methylation across the promoter regions of the dopamine receptor 4 gene (DRD4), the serotonin transporter gene (SLC6A4/SERT) and the X-linked monoamine oxidase A gene (MAOA) using DNA sampled at both ages 5 and 10 years in 46 MZ twin-pairs and 45 DZ twin-pairs (total n=182). Our data suggest that DNA methylation differences are apparent already in early childhood, even between genetically identical individuals, and that individual differences in methylation are not stable over time. Our longitudinal-developmental study suggests that environmental influences are important factors accounting for interindividual DNA methylation differences, and that these influences differ across the genome. The observation of dynamic changes in DNA methylation over time highlights the importance of longitudinal research designs for epigenetic research.

Serotonin synthesis, release and reuptake in terminals: a mathematical model.

BACKGROUND: Serotonin is a neurotransmitter that has been linked to a wide variety of behaviors including feeding and body-weight regulation, social hierarchies, aggression and suicidality, obsessive compulsive disorder, alcoholism, anxiety, and affective disorders. Full understanding of serotonergic systems in the central nervous system involves genomics, neurochemistry, electrophysiology, and behavior. Though associations have been found between functions at these different levels, in most cases the causal mechanisms are unknown. The scientific issues are daunting but important for human health because of the use of selective serotonin reuptake inhibitors and other pharmacological agents to treat disorders in the serotonergic signaling system. METHODS: We construct a mathematical model of serotonin synthesis, release, and reuptake in a single serotonergic neuron terminal. The model includes the effects of autoreceptors, the transport of tryptophan into the terminal, and the metabolism of serotonin, as well as the dependence of release on the firing rate. The model is based on real physiology determined experimentally and is compared to experimental data. RESULTS: We compare the variations in serotonin and dopamine synthesis due to meals and find that dopamine synthesis is insensitive to the availability of tyrosine but serotonin synthesis is sensitive to the availability of tryptophan. We conduct in silico experiments on the clearance of extracellular serotonin, normally and in the presence of fluoxetine, and compare to experimental data. We study the effects of various polymorphisms in the genes for the serotonin transporter and for tryptophan hydroxylase on synthesis, release, and reuptake. We find that, because of the homeostatic feedback mechanisms of the autoreceptors, the polymorphisms have smaller effects than one expects. We compute the expected steady concentrations of serotonin transporter knockout mice and compare to experimental data. Finally, we study how the properties of the the serotonin transporter and the autoreceptors give rise to the time courses of extracellular serotonin in various projection regions after a dose of fluoxetine. CONCLUSIONS: Serotonergic systems must respond robustly to important biological signals, while at the same time maintaining homeostasis in the face of normal biological fluctuations in inputs, expression levels, and firing rates. This is accomplished through the cooperative effect of many different homeostatic mechanisms including special properties of the serotonin transporters and the serotonin autoreceptors. Many difficult questions remain in order to fully understand how serotonin biochemistry affects serotonin electrophysiology and vice versa, and how both are changed in the presence of selective serotonin reuptake inhibitors. Mathematical models are useful tools for investigating some of these questions.

A PK2/Bv8/PROK2 antagonist suppresses tumorigenic processes by inhibiting angiogenesis in glioma and blocking myeloid cell infiltration in pancreatic cancer.

Infiltration of myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis in many types of cancer. The polypeptide chemokine PK2 (Bv8, PROK2) has been shown to regulate myeloid cell mobilization from the bone marrow, leading to activation of the angiogenic process, as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 were shown to display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. In this study we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the context of glioblastoma and pancreatic cancer xenograft tumor models. For the highly vascularized glioblastoma, PKRA7 was associated with decreased blood vessel density and increased necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 appears to be mediated by the blockage of myeloid cell migration/infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of certain pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both types of tumor. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by two distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy.

Coupling of beta2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes.

-Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (betaAR) signal transduction. Although several of these models have altered betaAR subtypes, the specific functional sequelae of betaAR stimulation in murine heart, particularly those of beta2-adrenergic receptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergic receptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-32P]GTP azidoanilide into alpha subunits of Gi2 and Gi3 after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gs and Gi signaling, resulting in null inotropic response, unless the Gi signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta2AR-coupled Gi proteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling.

Differential mechanisms of morphine antinociceptive tolerance revealed in (beta)arrestin-2 knock-out mice.

Morphine induces antinociception by activating mu opioid receptors (muORs) in spinal and supraspinal regions of the CNS. (Beta)arrestin-2 (beta)arr2), a G-protein-coupled receptor-regulating protein, regulates the muOR in vivo. We have shown previously that mice lacking (beta)arr2 experience enhanced morphine-induced analgesia and do not become tolerant to morphine as determined in the hot-plate test, a paradigm that primarily assesses supraspinal pain responsiveness. To determine the general applicability of the (beta)arr2-muOR interaction in other neuronal systems, we have, in the present study, tested (beta)arr2 knock-out ((beta)arr2-KO) mice using the warm water tail-immersion paradigm, which primarily assesses spinal reflexes to painful thermal stimuli. In this test, the (beta)arr2-KO mice have greater basal nociceptive thresholds and markedly enhanced sensitivity to morphine. Interestingly, however, after a delayed onset, they do ultimately develop morphine tolerance, although to a lesser degree than the wild-type (WT) controls. In the (beta)arr2-KO but not WT mice, morphine tolerance can be completely reversed with a low dose of the classical protein kinase C (PKC) inhibitor chelerythrine. These findings provide in vivo evidence that the muOR is differentially regulated in diverse regions of the CNS. Furthermore, although (beta)arr2 appears to be the most prominent and proximal determinant of muOR desensitization and morphine tolerance, in the absence of this mechanism, the contributions of a PKC-dependent regulatory system become readily apparent.

Beta-arrestin-mediated beta1-adrenergic receptor transactivation of the EGFR confers cardioprotection.

Deleterious effects on the heart from chronic stimulation of beta-adrenergic receptors (betaARs), members of the 7 transmembrane receptor family, have classically been shown to result from Gs-dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby beta-arrestins mediate beta1AR signaling to the EGFR. This beta-arrestin-dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein-coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via beta-arrestin-mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.

Anti-beta(1)-adrenergic receptor antibodies and heart failure: causation, not just correlation.

Antibodies specific for the beta(1)-adrenergic receptor are found in patients with chronic heart failure of various etiologies. From work presented in this issue of the JCI, we can now infer that these antibodies actually contribute to the pathogenesis of chronic heart failure. This commentary discusses mechanisms by which these antibodies may engender cardiomyopathy.

Enhanced rewarding properties of morphine, but not cocaine, in beta(arrestin)-2 knock-out mice.

The reinforcing and psychomotor effects of morphine involve opiate stimulation of the dopaminergic system via activation of mu-opioid receptors (muOR). Both mu-opioid and dopamine receptors are members of the G-protein-coupled receptor (GPCR) family of proteins. GPCRs are known to undergo desensitization involving phosphorylation of the receptor and the subsequent binding of beta(arrestins), which prevents further receptor-G-protein coupling. Mice lacking beta(arrestin)-2 (beta(arr2)) display enhanced sensitivity to morphine in tests of pain perception attributable to impaired desensitization of muOR. However, whether abrogating muOR desensitization affects the reinforcing and psychomotor properties of morphine has remained unexplored. In the present study, we examined this question by assessing the effects of morphine and cocaine on locomotor activity, behavioral sensitization, conditioned place preference, and striatal dopamine release in beta(arr2) knock-out (beta(arr2)-KO) mice and their wild-type (WT) controls. Cocaine treatment resulted in very similar neurochemical and behavioral responses between the genotypes. However, in the beta(arr2)-KO mice, morphine induced more pronounced increases in striatal extracellular dopamine than in WT mice. Moreover, the rewarding properties of morphine in the conditioned place preference test were greater in the beta(arr2)-KO mice when compared with the WT mice. Thus, beta(arr2) appears to play a more important role in the dopaminergic effects mediated by morphine than those induced by cocaine.

Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance.

Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [kappa opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4-L5 spinal dorsal horn of wild-type C57BL/6 mice (7-21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAP-positive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the kappa agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.

A genetically engineered thermally responsive sustained release curcumin depot to treat neuroinflammation.

Radiculopathy, a painful neuroinflammation that can accompany intervertebral disc herniation, is associated with locally increased levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Systemic administration of TNF antagonists for radiculopathy in the clinic has shown mixed results, and there is growing interest in the local delivery of anti-inflammatory drugs to treat this pathology as well as similar inflammatory events of peripheral nerve injury. Curcumin, a known antagonist of TNFα in multiple cell types and tissues, was chemically modified and conjugated to a thermally responsive elastin-like polypeptide (ELP) to create an injectable depot for sustained, local delivery of curcumin to treat neuroinflammation. ELPs are biopolymers capable of thermally-triggered in situ depot formation that have been successfully employed as drug carriers and biomaterials in several applications. ELP-curcumin conjugates were shown to display high drug loading, rapidly release curcumin in vitro via degradable carbamate bonds, and retain in vitro bioactivity against TNFα-induced cytotoxicity and monocyte activation with IC50 only two-fold higher than curcumin. When injected proximal to the sciatic nerve in mice via intramuscular (i.m.) injection, ELP-curcumin conjugates underwent a thermally triggered soluble-insoluble phase transition, leading to in situ formation of a depot that released curcumin over 4days post-injection and decreased plasma AUC 7-fold.