Ghrelin-mediated sympathoinhibition and suppression of inflammation in sepsis

Sepsis, a systemic inflammatory response to infection, continues to carry a high mortality despite advances in critical care medicine. Elevated sympathetic nerve activity in sepsis has been shown to contribute to early hepatocellular dysfunction and subsequently multiple organ failure, resulting in a poor prognosis, especially in the elderly. Thus, suppression of sympathetic nerve activity represents a novel therapeutic option for sepsis. Ghrelin is a 28-amino acid peptide shown to inhibit sympathetic nerve activity and inflammation in animal models of tissue injury. Age-related ghrelin hyporesponsiveness has also been shown to exacerbate sepsis. However, the mechanistic relationship between ghrelin-mediated sympathoinhibition and suppression of inflammation remains poorly understood. This review assesses the therapeutic potential of ghrelin in sepsis in the context of the neuroanatomical and molecular basis of ghrelin-mediated suppression of inflammation through inhibition of central sympathetic outflow.     

Immobilization of carbonic anhydrase in alginate and its influence on transformation of CO2 to calcite

Present investigation entails carbonic anhydrase (CA) immobilization and its influence on transformation of CO₂ to calcite. CA enzyme was immobilized in alginate beads, subsequently maintained its catalytic efficiency after sequential operational cycles. The immobilized beads showed better operational stability by retaining nearly 67% of its initial activity even after six cycles. Batch scale studies with free and immobilized enzyme revealed that the entrapped CA hydrates CO₂ to bicarbonate and/or carbonate which was then made to react with Ca²⁺ ions to transform into calcite. Calcite was characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The entrapped CA was employed for the performance evaluation with respect to several operational parameters including the influence of enzyme concentration in free and immobilized condition. It was concluded that immobilized CA in alginate beads would have the potential for CO₂ sequestration by biomimetic route.     

TREAT: a bioinformatics tool for variant annotations and visualizations in targeted and exome sequencing data

TREAT (Targeted RE-sequencing Annotation Tool) is a tool for facile navigation and mining of the variants from both targeted resequencing and whole exome sequencing. It provides a rich integration of publicly available as well as in-house developed annotations and visualizations for variants, variant-hosting genes and host-gene pathways. AVAILABILITY AND IMPLEMENTATION: TREAT is freely available to non-commercial users as either a stand-alone annotation and visualization tool, or as a comprehensive workflow integrating sequencing alignment and variant calling. The executables, instructions and the Amazon Cloud Images of TREAT can be downloaded at the website: http://ndc.mayo.edu/mayo/research/biostat/stand-alone-packages.cfm.     

Arthrobacter sp. lipase immobilization on magnetic sol–gel composite supports for enantioselectivity improvement

A bacterial lipase from Arthrobacter sp. (ABL: IIIM Jammu, India strain, MTCC No. 5125) has been immobilized on non-magnetic (Type A) and magnetic (Type B) supports derived from copolymerization of 3-aminopropyltriethoxysilane and tetraethylorthosilicate. Immobilized ABL presented 21–34 mg/g protein binding providing 30–75 units/g activity in Type A non-magnetic composites and 24–45 mg/g protein binding providing 35–90 units/g activity with Type B supports containing magnetic particles. Immobilized ABL preparations have shown enhanced stability at pH 5–9 and temperature up to 70 °C whereas free ABL is unstable under these conditions. Improved hydrolytic conversion as well as enantioselectivity were observed with acyl fluoxetine intermediate (ethyl 3-hydroxy-3-phenylpropanoate alkyl acylates) and chiral auxiliaryacyl 1-phenyl ethanol using immobilized ABL derivatives (ee ～99%; 3–4-fold increase in E-values) as compared to ABL enzyme/cells (ee 93–98%). Introduction of magnetic particles in these supports has led to easier separation process with high product recovery yields.     

The C-terminal Src Inhibitory Kinase (Csk)-mediated Tyrosine Phosphorylation Is a Novel Molecular Mechanism to Limit P2X3 Receptor Function in Mouse Sensory Neurons

On sensory neurons, sensitization of P2X₃ receptors gated by extracellular ATP contributes to chronic pain. We explored the possibility that receptor sensitization may arise from down-regulation of an intracellular signal negatively controlling receptor function. In view of the structural modeling between the Src region phosphorylated by the C-terminal Src inhibitory kinase (Csk) and the intracellular C terminus domain of the P2X₃ receptor, we investigated how Csk might regulate receptor activity. Using HEK cells and the in vitro kinase assay, we observed that Csk directly phosphorylated the tyrosine 393 residue of the P2X₃ receptor and strongly inhibited receptor currents. On mouse trigeminal sensory neurons, the role of Csk was tightly controlled by the extracellular level of nerve growth factor, a known algogen. Furthermore, silencing endogenous Csk in HEK or trigeminal cells potentiated P2X₃ receptor responses, confirming constitutive Csk-mediated inhibition. The present study provides the first demonstration of an original molecular mechanism responsible for negative control over P2X₃ receptor function and outlines a potential new target for trigeminal pain suppression.ATP-activated P2X₃ receptors are expressed almost exclusively by mammalian sensory neurons to play an important role in the transduction of painful stimuli to the central nervous system (1). Activation of P2X₃ receptors by ATP released during acute and chronic pain is thought to send nociceptive signals to central pain-related networks (2). In view of the multitude of environmental stimuli normally reaching sensory terminals, the question then arises how inappropriate activation of P2X₃ receptors is normally prevented. This process may contribute to suppression of continuous pain sensation in conjunction with central synaptic inhibition.The molecular pathways triggered by algogenic substances and responsible for modulating P2X₃ receptor structure and function remain incompletely understood. This topic is of particular interest because it can provide original clues for novel approaches related to treat pain. The nerve growth factor, NGF,² is one of the most powerful endogenous substances which elicit pain and inflammation via the tyrosine kinase receptor TrkA (3). This neurotrophin stimulates an intracellular cascade that elicits PKC-dependent P2X₃ receptor phosphorylation with ensuing facilitation of receptor currents. Conversely, suppression of NGF signaling powerfully down-regulates P2X₃ receptor function (4). These observations are consistent with the raised NGF levels in acute or inflammatory pain conditions (3). The molecular mechanisms underlying these effects remain, however, unclear.A dynamic balance between tyrosine phosphorylation and dephosphorylation is a major factor controlling the activity of many neurotransmitter receptors (5). TrkA stimulation activates intracellular signaling including Src tyrosine kinases (6) that, in neurons, are important modulators of ligand-gated receptors like nicotinic (7), NMDA receptors (8), and TRPV1 receptors (9). All these receptors are involved in mediating various types of pain in the spinal cord and sensory ganglia. There is, however, no available data on the role of tyrosine phosphorylation on P2X₃ receptor function.The fundamental regulator of Src signaling is the C-terminal Src kinase (Csk) that blocks it via tyrosine phosphorylation (Tyr-527, Refs. 10, 11). We explored whether tyrosine phosphorylation might regulate P2X₃ receptors of sensory neurons by focusing on the P2X₃ C-terminal domain Tyr-393 residue, which is included in a region with significant similarity with the Csk-phosphorylating region of Src. Our data demonstrate that Csk activation induced an increased tyrosine (Tyr-393 residue) P2X₃ receptor phosphorylation with decreased receptor function, observed both in mouse trigeminal sensory neurons as well as a cell expression system. We, thus, propose that Csk-mediated P2X₃ receptor inhibition is a novel mechanism to limit overactivation of P2X₃ receptors.     

Insights into the nature of Mo(V) species in solution: Modeling catalytic cycles for molybdenum enzymes

The tris(pyrazolyl)borate and related tripodal N-donor ligands originally developed by Trofimenko stabilize mononuclear compounds containing Mo^VIO₂, Mo^VIO, Mo^VO, and Mo^IVO units and effectively inhibit their polynucleation in organic solvents. Dioxo-Mo(VI) complexes of the type LMoO₂(SPh), where L = hydrotris(3,5-dimethylpyrazol-1-yl)borate (Tp^∗), hydrotris(3-isopropylpyrazol-1-yl)borate (Tp^iPr), and hydrotris(3,5-dimethyl-1,2,4-triazol-1-yl)borate (Tz) and related derivatives are the only model systems that mimic the complete reaction sequence of sulfite oxidase, in which oxygen from water is ultimately incorporated into product. The quasi-reversible, one-electron reduction of Tp^∗MoO₂(SPh) in acetonitrile exhibits a positive potential shift upon addition of a hydroxylic proton donor, and the magnitude of the shift correlates with the acidity of the proton donor. These reductions produce two Mo(V) species, [Tp^∗Mo^VO₂(SPh)]⁻ and Tp^∗Mo^VO(OH)(SPh), that are related by protonation. Measurement of the relative amounts of these two Mo(V) species by EPR spectroscopy enabled the pK_a of the Mo^V(OH) unit in acetonitrile to be determined and showed it to be several pK_a units smaller than that for water in acetonitrile. Similar electrochemical-EPR experiments for Tp^iPrMoO₂(SPh) indicated that the pK_a for its Mo^V(OH) unit was ∼1.7 units smaller than that for Tp^∗Mo^VO(OH)(SPh). Density functional theory calculations also predict a smaller pK_a for Tp^iPrMo^VO(OH)(SPh) compared to Tp^∗Mo^VO(OH)(SPh). Analysis of these results indicates that coupled electron-proton transfer (CEPT) is thermodynamically favored over the indirect process of metal reduction followed by protonation. The crystal structure of Tp^iPrMoO₂(SPh) is also presented.     

Fractionated X-radiation treatment can elicit an inducible-like radioprotective response that is not dependent on the intrinsic cellular X-radiation resistance/sensitivity

Inducible responses are well documented to play a role in the radiation response of cells. However, it is not known whether clinically relevant fractionated X-radiation treatment could elicit an inducible-like radioprotective response and whether there is a direct correlation between the inducible radiation response phenomenon and the intrinsic radiation response of the cell. Therefore, the purpose of this study was to determine whether closely related human colorectal tumor (HCT116) clones treated with fractionated X rays could elicit an inducible-like radiation response to a subsequent acute (i.e. single) X-ray challenge, and whether the magnitude of the inducible-like response correlates with the intrinsic X-ray resistance of the responding clones. After fractionated X irradiation, only the radiosensitive clone showed enhanced clonogenic survival with a subsequent acute X-ray exposure. Cell cycle changes or the selection of subclones with increased intrinsic radiation resistance induced by the fractionated X rays were excluded as the basis of this enhanced tolerance, suggesting the presence of an inducible-like radioprotective response. Using the comet assay, we found similar amounts of intrinsic DNA damage among the clones after acute X irradiation. Our findings demonstrate that fractionated X-ray treatment can elicit an inducible-like radioprotective response and represent the first evidence that this response is independent of the intrinsic radiation resistance/sensitivity of the responding cells.