Thiol-ene micropillar array electrospray ionization platform for zeptomole level bioanalysis.

A micropillar array electrospray ionization (µPESI) platform fabricated from thiol-enes with 56 individual polyethylene glycol coated µPESI chips for bioanalytical mass spectrometry is introduced. Bioanalysis capability is shown by measurement of a protein, a protein digest and a cell lysate sample. The thiol-ene polyethylene glycol (PEG) coated µPESI chip allows the use of a wide range of aqueous-organic solvent compositions and provides a detection limit at 60 zeptomole level (6 × 10-20 mol) for a peptide standard.

Fabrication of concave micromirrors for single cell imaging via controlled over-exposure of organically modified ceramics in single step lithography.

This work describes the fabrication of concave micromirrors to improve the sensitivity of fluorescence imaging, for instance, in single cell analysis. A new approach to fabrication of tunable round (concave) cross-sectional shaped microchannels out of the inorganic-organic hybrid polymer, Ormocomp®, via single step optical lithography was developed and validated. The concave micromirrors were implemented by depositing and patterning thin films of aluminum on top of the concave microchannels. The round cross-sectional shape was due to residual layer formation, which is inherent to Ormocomp® upon UV exposure in the proximity mode. We show that it is possible to control the residual layer thickness and thus the curved shape of the microchannel cross-sectional profile and eventually the focal length of the micromirror, by simply adjusting the UV exposure dose and the distance of the proximity gap (to the photomask). In general, an increase in the exposure dose or in the distance of the proximity gap results in a thicker residual layer and thus an increase in the radius of the microchannel curvature. Under constant exposure conditions, the radius of curvature is almost linearly dependent on the microchannel aspect ratio, i.e., the width (here, 20-200 µm) and the depth (here, 15-45 µm). Depending on the focal length, up to 8-fold signal enhancement over uncoated, round Ormocomp® microchannels was achieved in single cell imaging with the help of the converging micromirrors in an epifluorescence microscopy configuration.

Robust hybrid elastomer/metal-oxide superhydrophobic surfaces.

We introduce a new type of hybrid material: a nanostructured elastomer covered by a hard photoactive metal-oxide thin film resembling the exoskeleton of insects. It has extreme water repellency and fast self-recovery after damage. A new fabrication method for replicating high aspect ratio, hierarchical re-entrant aluminum structures into polydimethylsiloxane (PDMS) is presented. The method is based on a protective titania layer deposited by atomic layer deposition (ALD) on the aluminum template. The ALD titania transfers to the elastomeric scaffold via sacrificial release etching. The sacrificial release method allows for high aspect ratio, even 100 µm deep and successful release of overhanging structures, unlike conventional peeling. The ALD titania conformally covers the 3D multihierarchical structures of the template and protects the polymer during the release etch. Afterwards it prevents the high aspect ratio nanostructures from elasticity based collapse. The resulting nanostructured hybrid PDMS/titania replicas display robust superhydrophobicity without any further fluoro-coating or modification. Their mechanical and thermal robustness results from a thick nanostructured elastomeric layer which is conformally covered by ceramic titania instead of a monolayer hydrophobic coating. We have demonstrated the durability of these replicas against mechanical abrasion, knife scratches, rubbing, bending, peel tape test, high temperature annealing, UV exposure, water jet impingement and long term underwater storage. Though the material loses its superhydrophobicity in oxygen plasma exposure, a fast recovery from superhydrophilic to superhydrophobic can be achieved after 20 min UV irradiation. UV-assisted recovery is correlated with the high photoactivity of ALD titania film. This novel hybrid material will be applicable to the large area superhydrophobic surfaces in practical outdoor applications.

Pregabalin enhances the antinociceptive effect of oxycodone and morphine in thermal models of nociception in the rat without any pharmacokinetic interactions.

BACKGROUND: Oxycodone is increasingly being used in combination with pregabalin. Pregabalin use is prevalent in opioid-dependent individuals. A high number of deaths caused by the co-use of gabapentinoids and opioids occur. It is not known whether pregabalin affects concentrations of oxycodone or morphine in the central nervous system. METHODS: Effects of pregabalin on acute oxycodone or morphine-induced antinociception, tolerance and sedation were studied using tail-flick, hot plate and rotarod tests in male Sprague-Dawley rats. Concentrations of pregabalin, opioids and their major metabolites in the brain were quantified by mass spectrometry. RESULTS: In the hot plate test, morphine (2.5 mg/kg, s.c.) caused antinociception of 28% maximum possible effect (MPE), whereas pregabalin (50 mg/kg, i.p.) produced 8-10% MPE. Co-administration of pregabalin and morphine resulted in antinociception of 63% MPE. Oxycodone (0.6 mg/kg s.c.) produced antinociception of 18% MPE, which increased to 39% MPE after co-administration with pregabalin. When pregabalin 10 mg/kg was administered before oxycodone (0.6 mg/kg, s.c.) or morphine (2.5 mg/kg), only the effect of oxycodone was potentiated in the tail-flick and the hot plate tests. Brain concentrations of the opioids, their major metabolites and pregabalin were unchanged. Pregabalin co-administration (50 mg/kg, i.p., once daily) did not prevent the development of morphine tolerance. CONCLUSIONS: Pregabalin potentiated antinociceptive and sedative effects of oxycodone and morphine in acute nociception. Co-administration of pregabalin with the opioids did not affect the brain concentrations of oxycodone or morphine. Pregabalin did not prevent morphine tolerance.

Ketamine coadministration attenuates morphine tolerance and leads to increased brain concentrations of both drugs in the rat.

BACKGROUND AND PURPOSE: The effects of ketamine in attenuating morphine tolerance have been suggested to result from a pharmacodynamic interaction. We studied whether ketamine might increase brain morphine concentrations in acute coadministration, in morphine tolerance and morphine withdrawal. EXPERIMENTAL APPROACH: Morphine minipumps (6 mg·day(-1) ) induced tolerance during 5 days in Sprague-Dawley rats, after which s.c. ketamine (10 mg·kg(-1) ) was administered. Tail flick, hot plate and rotarod tests were used for behavioural testing. Serum levels and whole tissue brain and liver concentrations of morphine, morphine-3-glucuronide, ketamine and norketamine were measured using HPLC-tandem mass spectrometry. KEY RESULTS: In morphine-naïve rats, ketamine caused no antinociception whereas in morphine-tolerant rats there was significant antinociception (57% maximum possible effect in the tail flick test 90 min after administration) lasting up to 150 min. In the brain of morphine-tolerant ketamine-treated rats, the morphine, ketamine and norketamine concentrations were 2.1-, 1.4- and 3.4-fold, respectively, compared with the rats treated with morphine or ketamine only. In the liver of morphine-tolerant ketamine-treated rats, ketamine concentration was sixfold compared with morphine-naïve rats. After a 2 day morphine withdrawal period, smaller but parallel concentration changes were observed. In acute coadministration, ketamine increased the brain morphine concentration by 20%, but no increase in ketamine concentrations or increased antinociception was observed. CONCLUSIONS AND IMPLICATIONS: The ability of ketamine to induce antinociception in rats made tolerant to morphine may also be due to increased brain concentrations of morphine, ketamine and norketamine. The relevance of these findings needs to be assessed in humans.

The mineralocorticoid receptor antagonist spironolactone enhances morphine antinociception.

BACKGROUND: Spironolactone, a commonly used mineralocorticoid receptor antagonist, has been reported to potentiate the effect of morphine in the rat. The aim of this study was to investigate the effects of spironolactone on morphine antinociception and tissue distribution. METHODS: The effects of spironolactone on acute morphine-induced antinociception, induction of morphine tolerance and established morphine tolerance were studied with tail-flick and hot plate tests in male Sprague-Dawley rats. Serum, brain, and liver morphine and its metabolite concentrations were quantified using high-pressure liquid chromatography-tandem mass spectrometry. Spironolactone was also administered with the peripherally acting, P-glycoprotein (P-gp) substrate loperamide to test whether spironolactone allows loperamide to pass the blood-brain barrier. RESULTS: Spironolactone (50 mg/kg, i.p.) had no antinociceptive effects of its own, but it enhanced the antinociceptive effect of morphine in both thermal tests. Two doses of spironolactone enhanced the maximum possible effect (MPE) from 19.5% to 100% in the hot plate test 90 min after administration of 4 mg/kg morphine. Morphine concentrations in the brain were increased fourfold at 90 min by spironolactone. Spironolactone did not inhibit the formation of morphine-3-glucuronide. Acute spironolactone restored morphine antinociception in morphine-tolerant rats but did not inhibit the development of tolerance. The peripherally restricted opioid, loperamide (10 mg/kg), had no antinociceptive effects when administered alone, but co-administration with spironolactone produced a 40% MPE in the hot plate test. CONCLUSIONS: Spironolactone has no antinociceptive effects in thermal models of pain, but it enhances the antinociceptive effects of morphine mainly by increasing morphine central nervous system concentrations, probably by inhibiting P-gp.

UV-embossed inorganic-organic hybrid nanopillars for bioapplications.

We demonstrate rapid and maskless wafer-scale fabrication of inorganic-organic hybrid nanopillars for microfluidic and mass spectrometric applications. The nanopillar features are transferred from a black silicon master to the hybrid material by UV-embossing with a composite stamp made of polydimethylsiloxane (PDMS)/hard-PDMS (h-PDMS), which allows accurate transfer of structures in the 100 nm size range. The nanopillar embossing process is extremely simple and well suited for cheap wafer-scale prototyping, as the process does not require expensive masks or stamps and even works with a standard office lamp as the exposure source. The pillars can also be fabricated on a wide variety of substrates because the hybrid material is solvent-free. The use of the hybrid nanopillars as a hydrophobic/philic platforms and as sample plates in surface assisted laser desorption ionization mass spectrometry is demonstrated.

Temporal age-related changes in spectral, fractal and complexity characteristics of heart rate variability.

Cross-sectional studies have suggested that heart rate (HR) variability, analysed using traditional time and frequency domain methods, is related to ageing, but no longitudinal studies have estimated the age dependence of HR fluctuation. This study evaluated temporal age-related changes in 12-h measures of HR variability among 109 patients with coronary artery disease (CAD), who underwent repeat Holter recordings at 32-month intervals. Time and frequency domain measures, along with fractal and complexity measures of HR variability, were determined at the baseline and after 32 months. Changes in HR dynamics were compared with various laboratory variables, exercise data and angiographic progression of CAD. Traditional time and frequency domain measures of HR variability did not change significantly during the follow-up, but the power-law scaling slope decreased from -1.29 +/- 0.20 to -1.36 +/- 0.23 (P<0.01) and the short-term fractal exponent (alpha1) of HR dynamics from 1.29 +/- 0.14-1.22 +/- 0.18 (P<0.001). The approximate entropy value also decreased from 1.00 +/- 0.19 to 0.95 +/- 0.18 (P<0.05). The changes in HR behaviour were not related to demographic data, laboratory values or angiographic progression of CAD. Only a weak correlation was observed between the change in the power-law slope and the baseline glucose value (P<0.05). This longitudinal study shows that the fractal characteristics of HR dynamics and the complexity properties of R-R intervals undergo rapid changes along with ageing, and that fractal and complexity analysis techniques are more sensitive than traditional analysis methods in documenting temporal age-related changes in HR behaviour.

Heart rate variability and progression of coronary atherosclerosis.

Low heart rate (HR) variability is associated with increased risk of cardiovascular morbidity and mortality, but the causes and mechanisms of this association are not well known. This prospective study was designed to test the hypothesis that reduced HR variability is related to progression of coronary atherosclerosis. Average HR and HR variability were analyzed in 12-hour ambulatory ECG recordings from 265 qualified patients participating in a multicenter study to evaluate the angiographic progression of coronary artery disease in patients with prior coronary artery bypass surgery and low high-density lipoprotein cholesterol concentrations (<1.1 mmol/L). Participants were randomized to receive a placebo or gemfibrozil therapy. The progression of coronary atherosclerosis was estimated by quantitative, computer-assisted analysis of coronary artery stenoses from the baseline angiograms and from repeated angiograms performed an average of 32 months later. The progression of focal coronary atherosclerosis of the patients randomized to placebo therapy was more marked in the tertile with the lowest standard deviation of all normal to normal R-R intervals (SDNN, 74+/-13 ms; mean decrease in the per-patient minimum luminal diameter -0.17 mm; 95% confidence interval [CI], -0.23 to -0.12 mm) than in the middle tertile (SDNN, 107+/-7 ms; mean decrease -0.05 mm; 95% CI, -0.08 to -0.01 mm) or highest tertile (SDNN, 145+/-25 ms; mean change 0.01 mm; 95% CI, -0. 04 to 0.02 mm) (P<0.001 between the tertiles). This association was abolished by gemfibrozil. SDNN was lower (P<0.001) and minimum HR was faster (P<0.01) in the patients with marked progression than in those with regression of focal coronary atherosclerosis. In multiple regression analysis including HR variability, minimum HR, demographic and clinical variables, smoking, blood pressure, glucose, lipid measurements and lipid-modifying therapy, progression of focal coronary atherosclerosis was independently predicted by the SDNN (beta=0.24; P=0.0001). Low HR variability analyzed from ambulatory ECG predicts rapid progression of coronary artery disease. HR variability provided information on progression of focal coronary atherosclerosis beyond that obtained by traditional risk markers of atherosclerosis.