Relationship between membrane phosphatidylinositol-4,5-bisphosphate and receptor-mediated inhibition of native neuronal M channels

The relationship between receptor-induced membrane phosphatidylinositol-4'5'-bisphosphate (PIP2) hydrolysis and M-current inhibition was assessed in single-dissociated rat sympathetic neurons by simultaneous or parallel recording of membrane current and membrane-to-cytosol translocation of the fluorescent PIP2/inositol 1,4,5-trisphosphate (IP3)-binding peptide green fluorescent protein-tagged pleckstrin homology domain of phospholipase C (GFP-PLC delta-PH). The muscarinic receptor agonist oxotremorine-M produced parallel time- and concentration-dependent M-current inhibition and GFP-PLC delta-PH translocation; bradykinin also produced parallel time- dependent inhibition and translocation. Phosphatidylinositol-4-phosphate-5-kinase (PI5-K) overexpression reduced both M-current inhibition and GFP-PLC delta-PH translocation by both oxotremorine-M and bradykinin. These effects were partly reversed by wortmannin, which inhibits phosphatidylinositol-4-kinase (PI4-K). PI5-K overexpression also reduced the inhibitory action of oxotremorine-M on PIP2-gated G-protein-gated inward rectifier (Kir3.1/3.2) channels; bradykinin did not inhibit these channels. Overexpression of neuronal calcium sensor-1 protein (NCS-1), which increases PI4-K activity, did not affect responses to oxotremorine-M but reduced both fluorescence translocation and M-current inhibition by bradykinin. Using an intracellular IP3 membrane fluorescence-displacement assay, initial mean concentrations of membrane [PIP2] were estimated at 261 mu M (95% confidence limit; 192-381 mu M), rising to 693 mu M (417-1153 mu M) in neurons overexpressing PI5-K. Changes in membrane [PIP2] during application of oxotremorine-M were calculated from fluorescence data. The results, taken in conjunction with previous data for KCNQ2/3 (Kv7.2/Kv7.3) channel gating by PIP2 (Zhang et al., 2003), accorded with the hypothesis that the inhibitory action of oxotremorine-M on M current resulted from depletion of PIP2. The effects of bradykinin require additional components of action, which might involve IP3-induced Ca2+ release and consequent M-channel inhibition (as proposed previously) and stimulation of PIP2 synthesis by Ca2+-dependent activation of NCS-1

Cats, comics and Knausgård : promoting student reading at a UK academic library with a leisure reading collection

This case study describes the creation of a leisure reading collection in the Clifford Whitworth library at the University of Salford. It briefly surveys existing literature on leisure reading collections and looks at the growing interest among UK academic libraries in recreational reading. It considers the reasons for promoting reading as a leisure activity to students and describes the processes of selecting, purchasing and marketing the collection at Salford. It also considers possible future developments for the collection and the evaluation of the library’s attempts to encourage a culture of reading amongst Salford students. The positive response to the collection suggests the development of leisure reading is a worthwhile activity for academic libraries to focus on and the study contains useful information for others who are interested in creating a similar collection

The role of information in nonstationary regression

The role of standard likelihood based measures of information and efficiency is unclear when regressions involve nonstationary data. Typically the standardized score is not asymptotically Gaussian and the standardized Hessian has a stochastic, rather than deterministic limit. Here we consider a time series regression involving a deterministic covariate which can be evaporating, slowly evolving or nonstationary. It is shown that conditional information, or equivalently, profile Kullback-Leibler and Fisher Information remain informative about both the accuracy, i.e. asymptotic variance, of profile maximum likelihood estimators, as well as the power of point optimal invariant tests for a unit root. Specifically these information measures indicate fractional, rather than linear trends may minimize inferential accuracy. Such is confirmed in numerical experiment

Hepatic resection for metastatic colorectal adenocarcinoma: A proposal of a prognostic scoring system

Background: Hepatic resection for metastatic colorectal cancer provides excellent longterm results in a substantial proportion of patients. Although various prognostic risk factors have been identified, there has been no dependable staging or prognostic scoring system for metastatic hepatic tumors. Study Design: Various clinical and pathologic risk factors were examined in 305 consecutive patients who underwent primary hepatic resections for metastatic colorectal cancer. Survival rates were estimated by the Cox proportional hazards model using the equation: S(t) = [S(o)(t)](exp(R - R(o))), where S(o)(t) is the survival rate of patients with none of the identified risk factors and R(o) = 0. Results: Preliminary multivariate analysis revealed that independently significant negative prognosticators were: (1) positive surgical margins, (2) extrahepatic tumor involvement including the lymph node(s), (3) tumor number of three or more, (4) bilobar tumors, and (5) time from treatment of the primary tumor to hepatic recurrence of 30 months or less. Because the survival rates of the 62 patients with positive margins or extrahepatic tumor were uniformly very poor, multivariate analysis was repeated in the remaining 243 patients who did not have these lethal risk factors. The reanalysis revealed that independently significant poor prognosticators were: (1) tumor number of three or more, (2) tumor size greater than 8 cm, (3) time to hepatic recurrence of 30 months or less, and (4) bilobar tumors. Risk scores (R) for tumor recurrence of the culled cohort (n = 243) were calculated by summation of coefficients from the multivariate analysis and were divided into five groups: grade 1, no risk factors (R = 0); grade 2, one risk factor (R = 0.3 to 0.7); grade 3, two risk factors (R = 0.7 to 1.1); grade 4, three risk factors (R = 1.2 to 1.6); and grade 5, four risk factors (R > 1.6). Grade 6 consisted of the 62 culled patients with positive margins or extrahepatic tumor. Kaplan-Meier and Cox proportional hazards estimated 5-year survival rates of grade 1 to 6 patients were 48.3% and 48.3%, 36.6% and 33.7%, 19.9% and 17.9%, 11.9% and 6.4%, 0% and 1.1%, and 0% and 0%, respectively (p < 0.0001). Conclusions: The proposed risk-score grading predicted the survival differences extremely well. Estimated survival as determined by the Cox proportional hazards model was similar to that determined by the Kaplan-Meier method. Verification and further improvements of the proposed system are awaited by other centers or international collaborative studies

Kv7/M-type potassium channels in rat skin keratinocytes.

Skin keratinocytes fulfil important signalling and protective functions. Immunocytochemical experiments revealed the unexpected presence of immunoreactivity for the M-type potassium channel subunit Kv7.2 in the keratinocyte layer of intact rat paw skin and in keratinocytes isolated from the skin of 1-day-old rats and cultured in vitro for 3-10 days. Application of the M-channel enhancer retigabine (3-10 μM) to isolated cultured rat keratinocytes: (a) increased outward membrane currents recorded under voltage clamp, (b) produced ~3 mV hyperpolarization at rest, (c) enhanced ~3-fold the release of ATP induced by the TRPV3 agonist carvacrol (1 mM) and (d) increased the amplitude of the carvacrol-induced intracellular Ca(2+) transient measured with Fura-2. The effect of retigabine on ATP release was prevented by the M-channel blocking agent XE991. We conclude that rat skin keratinocytes possess M-channels that, when activated, can modify their physiological properties, with potential significance for their sensory and other biological functions

KCNQ/M currents in sensory neurons: Significance for pain therapy

Neuronal hyperexcitability is a feature of epilepsy and both inflammatory and neuropathic pain. M currents [I-K(M)] play a key role in regulating neuronal excitability, and mutations in neuronal KCNQ2/3 subunits, the molecular correlates of I-K(M), have previously been linked to benign familial neonatal epilepsy. Here, we demonstrate that KCNQ/M channels are also present in nociceptive sensory systems. I-K(M) was identified, on the basis of biophysical and pharmacological properties, in cultured neurons isolated from dorsal root ganglia (DRGs) from 17-d-old rats. Currents were inhibited by the M-channel blockers linopirdine (IC50, 2.1 muM) and XE991 (IC50, 0.26 muM) and enhanced by retigabine (10 muM). The expression of neuronal KCNQ subunits in DRG neurons was confirmed using reverse transcription-PCR and single-cell PCR analysis and by immunofluorescence. Retigabine, applied to the dorsal spinal cord, inhibited C and Adelta fiber-mediated responses of dorsal horn neurons evoked by natural or electrical afferent stimulation and the progressive "windup" discharge with repetitive stimulation in normal rats and in rats subjected to spinal nerve ligation. Retigabine also inhibited responses to intrapaw application of carrageenan in a rat model of chronic pain; this was reversed by XE991. It is suggested that I-K(M) plays a key role in controlling the excitability of nociceptors and may represent a novel analgesic target

Prospects of oral disease control in the future - an opinion

The mouth supports a diverse microbiota which provides major benefits to the host. On occasions, this symbiotic relationship breaks down (dysbiosis), and disease can be a consequence. We argue that progress in the control of oral diseases will depend on a paradigm shift away from approaches that have proved successful in medicine for many diseases with a specific microbial aetiology. Factors that drive dysbiosis in the mouth should be identified and, where possible, negated, reduced or removed, while antimicrobial agents delivered by oral care products may function effectively, even at sub-lethal concentrations, by modulating the activity and growth of potentially pathogenic bacteria. In this way, the beneficial activities of the resident oral microbiota will be retained and the risk of dysbiosis occurring will be reduced

Stimulated emission depletion following two photon excitation

The technique of stimulated emission depletion of fluorescence (STED) from a two photon excited molecular population is demonstrated in the S, excited state of fluorescein in ethylene glycol and methanol. Two photon excitation (pump) is achieved using the partial output of a regeneratively amplified Ti:Sapphire laser in conjunction with an optical parametric amplifier whose tuneable output provides a synchronous depletion (dump) pulse. Time resolved fluorescence intensity and anisotropy measurements of the fluorescein emission are made using picosecond time-correlated single photon counting. Pump-dump time delayed fluorescence intensity measurements are used to characterise the response of the system and to provide additional data on saturation dynamics of the dump transition. Two photon STED is modelled using both approximate analytical techniques in the weak dump limit and by numerical solutions to the appropriate rate equations. The latter are used to fit experimental data from which it is possible to determine the cross-section for the stimulated transition and lifetime of the upper vibrational levels of the ground state

Polarised stimulated emission depletion studies of two-photon excited states

Stimulated emission depletion (STED) population and polarisation dynamics following two-photon excitation are investigated for rhodamine 6G in ethylene glycol. Time resolved fluorescence intensity and polarisation measurements were made using picosecond time-correlated single photon counting (TCSPC). Cross-sections for the stimulated transition were measured between 614nm (2.32 X 10(-16) cm(2)) and 663.5nm (6.05 X 10(-17) cm(2)), ground state vibrational lifetimes were found to vary between 314fs and 467fs. A collinear (180degrees) excitation-detection geometry was employed to investigate re-polarisation of the excited state array yielding fluorescence anisotropies above the two-photon limit. The circumvention of single-photon selection rules is demonstrated allowing the measurement of higher order parameters and correlation functions that are wholly inaccessible to 'conventional' (spontaneous) time resolved fluorescence techniques

Dental plaque as a biofilm and a microbial community – implications for health and disease

Dental plaque is a structurally- and functionally-organized biofilm. Plaque forms in an ordered way and has a diverse microbial composition that, in health, remains relatively stable over time (microbial homeostasis). The predominant species from diseased sites are different from those found in healthy sites, although the putative pathogens can often be detected in low numbers at normal sites. In dental caries, there is a shift toward community dominance by acidogenic and acid-tolerating species such as mutans streptococci and lactobacilli, although other species with relevant traits may be involved. Strategies to control caries could include inhibition of biofilm development (e.g. prevention of attachment of cariogenic bacteria, manipulation of cell signaling mechanisms, delivery of effective antimicrobials, etc.), or enhancement of the host defenses. Additionally, these more conventional approaches could be augmented by interference with the factors that enable the cariogenic bacteria to escape from the normal homeostatic mechanisms that restrict their growth in plaque and out compete the organisms associated with health. Evidence suggests that regular conditions of low pH in plaque select for mutans streptococci and lactobacilli. Therefore, the suppression of sugar catabolism and acid production by the use of metabolic inhibitors and non-fermentable artificial sweeteners in snacks, or the stimulation of saliva flow, could assist in the maintenance of homeostasis in plaque. Arguments will be presented that an appreciation of ecological principles will enable a more holistic approach to be taken in caries control