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Objective: To compare the indications and efficacy of endoscopic over‐under tympanoplasty versus endoscopic underlay tympanoplasty. Methods: Retrospective cohort study of patients undergoing type I endoscopic tympanoplasty via either an underlay or over‐under technique by a single surgeon from 2017 to 2021. Patients were excluded if they had a concurrent mastoidectomy, ossiculoplasty, or advanced cholesteatoma defined by involvement of multiple subsites. Patient demographics, perforation size and location, middle ear status, preoperative and postoperative audiograms, and perforation closure were reviewed. Middle ear status was represented using the Ossiculoplasty Outcome Parameter Score (OOPS). The primary outcome was perforation closure at most recent follow‐up and secondary outcomes were change in postoperative pure‐tone average (PTA) and air‐bone gap (ABG). Results: Of 48 patients, 27 underwent endoscopic underlay tympanoplasty and 21 underwent endoscopic over‐under tympanoplasty. Tragal cartilage‐perichondrium graft was used in 90% of procedures. Distribution of OOPS scores was not significantly different between groups. Over‐ under technique addressed significantly larger perforations (mean size of 54% vs. 31%, p <.001) and a higher rate of anterior extension (95% vs. 22%, p <.001) than underlay technique. Perforation closure rate was not different between groups (95% vs. 96%). Patients experienced significant improvement in PTA and ABG in both groups. Conclusion: The endoscopic over‐under tympanoplasty is comparable to endoscopic underlay tympanoplasty in terms of graft take and audiologic improvement. The over‐under technique is effective for repairing larger perforations or those with anterior extension. Level of evidence: IV [ABSTRACT FROM AUTHOR]

A novel sulfopropyl ether γ-cyclodextrin polymer (SPE-γ-CDP) through polycondensating sulfated cyclodextrins (SCDs) was synthesized. This synthesis approach also has the potential of preparing other derived cyclodextrins (CDs) polymers. The polymerized SCDs took on both the properties of SCDs and certain characteristics of polymers, such as chiral selectivity and high viscosity. Synthesis parameters, including reactions sequence, sulfation, and polycondensation conditions were investigated systematically. The product was characterized by elemental analysis, infrared spectroscopy (IR), and indirect UV detections prior to use as background electrolytes additive. The separation conditions, including the concentration of SPE-γ-CDP, the concentration and pH of running buffer, separation voltage, as well as the additional organic solution were optimized during chiral separation of neutral, acidic, and basic enantiomers in capillary electrophoresis (CE). SPE-γ-CDP was proven to be an effective chiral resolving agent in CE with the advantages of simple synthesis process, low cost, similar ratio of charge-to-mass, low current, great reproducibility, and reusability. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Objectives. To evaluate the evidence base for the content of initiatives that the alcohol industry implemented to reduce drink driving from 1982 to May 2015. Methods. We systematically analyzed the content of 266 global initiatives that the alcohol industry has categorized as actions to reduce drink driving. Results. Social aspects public relations organizations (i.e., organizations funded by the alcohol industry to handle issues that may be damaging to the business) sponsored the greatest proportion of the actions. Only 0.8% (n = 2) of the sampled industry actions were consistent with public health evidence of effectiveness for reducing drink driving. Conclusions. The vast majority of the alcohol industry's actions to reduce drink driving does not reflect public health evidenced-based recommendations, even though effective drink-driving countermeasures exist, such as a maximum blood alcohol concentration limit of 0.05 grams per deciliter for drivers and widespread use of sobriety checkpoints. [ABSTRACT FROM AUTHOR]

Background: Health is influenced by determinants beyond the traditional conception of the health sector. Increasingly, global actors are targeting policymakers at global and national levels to take an intersectoral approach to health issues. Multilateral organizations in the health and transport communities have published policy reports targeting policymakers to address the burden of road traffic injuries. However, these reports stem from sectors grounded in different disciplinary perspectives. We investigate whether sectors have differing evidentiary traditions by analyzing differences regarding author networks, type of evidence cited, recommendations, and indicators. Methods: We selected global policy reports on road traffic injury prevention based upon expert opinion and categorized them by sector according to their institutional publisher. For each report, we i) conducted an authorship analysis by sectoral affiliation; ii) analyzed the types of research evidence cited and categorized the evidence type and institutional nature of the publisher; iii) analyzed key recommendations by extracting recommendations presented in the concluding sections of the documents; and iv) examined the use of indicators. Descriptive statistics were used to determine whether dimensions differed by the sectoral affiliation of the policy report. Results: Authorship was dominated by the sector from which the report was published, while reports that involved both sectors often showed clustering of authors in one sector or another, depending on the subject addressed. Reports originating from different sectors preferentially cited different types of evidence; notably, health sector reports emphasized observational studies and reviews, while transport sector reports drew heavily on government agency reports. There were no differences in recommendations and indicators used. Conclusions: Notions of knowledge validity and valuations of evidence vary depending on the field's historical development. Such differences in valuing evidence within sectors may have the potential to undermine the application of evidence in intersectoral policymaking. Strategies to address this challenge include the identification of key individuals to connect separate sectors, knowledge translation activities that take account of sectoral differences, and the tailoring of messages to different audiences. Future analyses on other intersectoral issues may provide clarity on points of tension and differing types of evidence used in intersectoral work. [ABSTRACT FROM AUTHOR]

This paper describes the development of a novel high-throughput hollow fiber membrane solvent microextraction technique for the simultaneous measurement of the octanol/water distribution coefficient (log D ) for organic compounds such as drugs. The method is based on a designed system, which consists of a 96-well plate modified with 96 hollow fiber membrane tubes and a matching lid with 96 center holes and 96 side holes distributing in 96 grids. Each center hole was glued with a sealed on one end hollow fiber membrane tube, which is used to separate the aqueous phase from the octanol phase. A needle, such as microsyringe or automatic sampler, can be directly inserted into the membrane tube to deposit octanol as the accepted phase or take out the mixture of the octanol and the drug. Each side hole is filled with aqueous phase and could freely take in/out solvent as the donor phase from the outside of the hollow fiber membranes. The log D can be calculated by measuring the drug concentration in each phase after extraction equilibrium. After a comprehensive comparison, the polytetrafluoroethylene hollow fiber with the thickness of 210 μm, an extraction time of 300 min, a temperature of 25 °C and atmospheric pressure without stirring are selected for the high throughput measurement. The correlation coefficient of the linear fit of the log D values of five drugs determined by our system to reference values is 0.9954, showed a nice accurate. The −8.9% intra-day and −4.4% inter-day precision of log D for metronidazole indicates a good precision. In addition, the log D values of eight drugs were simultaneously and successfully measured, which indicated that the 96 throughput measure method of log D value was accurate, precise, reliable and useful for high throughput screening. [ABSTRACT FROM AUTHOR]

The article presents comments on a perioperative approach to the opioid crisis in Canada in 2018. It blames overprescribing for the increasing risk of adverse effects, dependence and opioid use disorders. The challenge of providing balanced analgesia after surgery facing clinicians is discussed. Possible solutions for addressing the opioid crisis are provided, including the use of predictive tools to individualize opioid prescriptions and improved community follow-ups.

Background: Despite the same surgical approach, up to 40% of patients develop chronic postsurgical pain (CPSP) following cardiac surgery, whereas the rest are chronic pain free. This variability suggests that CPSP is controlled partially through genetics, but the genes for CPSP are largely unknown. Aims: The aim of this study was to identify potential CPSP phenotypes by comparing patients who developed CPSP following cardiac surgery vs. those who did not. Methods: A research ethics board–approved, cross-sectional study of post–cardiac surgery pain was conducted at Toronto General Hospital from 2011 to 2015. Patients were recruited to complete a short survey of chronic pain scores and the Short-Form McGill Pain Questionnaire–2. A subset of patients completed a longer survey of eight validated pain phenotyping questionnaires and/or four psychophysical assessments. All surveys and psychophysical testing were conducted after surgery. Patients were stratified by presence of chronic pain and groups were compared using descriptive statistics. Results: Six hundred forty-three patients completed the short form survey. The mean postsurgery assessment time was 41.5 (SD = ±25.1) months. Over a quarter (27.8%) reported CPSP at the chest as a consequence of their surgery. Of patients reporting CPSP, 46.6% reported mild pain (0–3), 35.8% reported moderate pain (4–7), and 17.6% reported severe pain (7–10) in accordance with the numerical rating scale. Patients with moderate and/or severe CPSP were younger, had a greater body mass index, and had higher anxiety sensitivity, pain catastrophizing, and somatization scores. Conclusions: Chronic pain levels after cardiac surgery are associated with anxiety, catastrophizing, and sensory abnormalities in body parts outside the field innervated by injured nerves, indicating the presence of widespread central sensitization to incoming sensory inputs from intact nerves. [ABSTRACT FROM AUTHOR]

There is significant overlap in the pharmacological management of pain and psychological disorders. Appropriate treatment of patients' comorbid psychological disorders, including sleep disturbances often leads to an improvement in reported pain intensity. The three first line agents for neuropathic pain include tricyclic antidepressants and serotonin norepinephrine reuptake inhibitors which are medications originally developed as antidepressants. The other first line medication for chronic neuropathic pain are anticonvulsant medications initially brought to the market-place for the treatment of epilepsy and are also now being used for the treatment of anxiety disorders and substance withdrawal symptoms. The efficacy of opioids for chronic pain is contentious, but it is agreed that the patients at highest risk for opioid misuse and addiction are patients with underlying psychological disorders who use opioids for their euphoric effects. Similarly, benzodiazepines may present a problem in patients with chronic pain, as up to one third of patients with pain are concomitantly prescribed benzodiazepines, and when combined with other sedating analgesic medications they put patients at increased risk for adverse events and polysubstance misuse. Finally, there is growing evidence for the efficacy of cannabis for treating neuropathic pain, but the consumption of cannabis has been associated with increased risk of psychosis in adolescents, and may be associated with an increased risk for developing bipolar disorder and anxiety disorders. The use of cannabis is associated with an increased risk of substance misuse in both adolescents and adults. In this narrative review, we examine the evidence for the use of several medications used for the treatment of both pain and psychological disorders, and their proposed mechanisms of action, in addition to special concerns for patients with comorbid pain and psychological disorders. [ABSTRACT FROM AUTHOR]

Summary: A novel molecularly imprinted hydrogel (X‐MIP) was developed with poly (allylamine hydrochloride) as monomer and epichlorohydrin as crosslinker. After optimising preparation conditions including reaction temperature, the amount of NaOH and template xylose, the X‐MIP was characterised through Fourier transform infrared spectroscopy, scanning electron microscope and swelling ratio. Xylose and glucose were employed to evaluate the adsorption capacity and selectivity of the X‐MIP. Its adsorption to xylose followed pseudo‐second‐order rate kinetics and the adsorption process was multilayer adsorption based on the matched Freundlich isotherm model. Its adsorption capacity to xylose was up to 398.68 mg g−1. The similar adsorption abilities in five successive runs and the good desorption rate (98.9%) verified its reusability. The selectivity for xylose and glucose could reach 4.70, and their separation factors in simulated xylitol production feedstocks were 591.05 (xylose:glucose, 99:1) and 15.42 (xylose:glucose, 3:1), respectively. Results indicated the prepared X‐MIP has good potential in industrial xylose purification. [ABSTRACT FROM AUTHOR]

A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237–0.833 U/ml and 9.013–10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis . The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity. [ABSTRACT FROM AUTHOR]

A novel high-throughput sample pretreatment system was developed by the integration of protein precipitation (PP), phospholipid removal (PPR), and hollow fiber liquid-phase microextraction (HF-LPME) into two simple 96-well plates and a matching 96-grid lid. With this system, 16 steroids were separated from biological matrices of plasma, milk, and urine and analyzed by liquid chromatography-triple quadrupole mass spectrometry. In the tandem sample cleanup process, the prepositive PP and PPR step preliminarily removed some of the interferences from the biological matrices. The following HF-LPME step kept the residual interference out of the hollow fiber and enriched the steroids in the hollow fiber to achieve high sensitivity. By a series of method optimizations, acetonitrile was chosen as the crash solvent for PP and PPR. A mixture of octanol and toluene (1:1 v/v) was used as the acceptor phase for HF-LPME. The extraction was conducted at 80 rpm for 50 min in a donor phase containing 1 mL 20 % sodium chloride at 25 °C. Under these conditions, the limits of detection for the 16 steroids were 3.6-300.0 pgmL in plasma, 3.0-270.0 pgmL in milk, and 2.2-210.0 pgmL in urine. The recoveries of the 16 steroids were 81.9-97.9 % in plasma (relative standard deviation 1.0-8.0 %), 80.6-97.7 % in milk (relative standard deviation 0.8-5.4 %), and 87.3-98.7 % in urine (relative standard deviation 1.0-4.9 %). Further, the integrated 96-well platform of PP, PPR, and HF-LPME enabled us to run this assay in an automatic and high-throughput fashion. The reliability of the method was further corroborated by evaluation of its applicability in plasma and urine samples from volunteers and fresh bovine milk from local dairy enterprises. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A novel boronic acid and carboxyl‐modified glucose molecularly imprinted polymer were prepared through suspension polymerization, which is based on 1.0 mmol glucose as a template, 1.2 mmol methacrylamidophenylboronic acid, and 6.8 mmol methacrylic acids as monomers, 19 mmol ethyleneglycol dimethacrylate, and 1 mmol methylene‐bis‐acrylamide as crosslinkers. The prepared glucose‐molecularly imprinted polymer had a particle size of 25–70 μm, and was thermally stable below 215°C, with a specific surface area of 174.82 m2/g and average pore size of 9.48 nm. The best selectivity between glucose and fructose was 2.71 and the maximum adsorption capacity of glucose‐ molecularly imprinted polymer was up to 236.32 mg/g which was consistent with the Langmuir adsorption model. The similar adsorption abilities in six successive runs and the good desorption rate (99.4%) verified glucose‐molecularly imprinted polymer could be reused. It was successfully used for extracting glucose from cellulose hydrolysis. The adsorption amount of glucose was 2.61 mg/mL and selectivity between glucose and xylose reached 4.12. A newly established chromatography (glucose‐molecularly imprinted polymer) mediated hollow fiber membrane method in time separated pure glucose from cellulose hydrolysates on a large scale, and purified glucose solution with a concentration of 3.84 mg/mL was obtained, which offered a feasible way for the industrial production of glucose from cellulose hydrolysates. [ABSTRACT FROM AUTHOR]

Abstract: An effective, rapid and reliable capillary electrophoresis–laser induced fluorescence (CE–LIF) procedure was built to study the characterization of tyrosine kinase (TK), which was a target for drug screening. In this procedure, CE separated the sample of the TK reaction and LIF selectively detected the fluorescence-labeled polypeptide substrate and product. The precise TK activity was quantitated by introducing the transformation ratio of the substrate (T%) to avoid the deviation resulted from the detection sensitivity and the injection amounts in different runs and different capillaries. By measuring the T%, the effects of various reaction conditions were optimized. Meanwhile, the progression of the enzyme reaction was monitored. The K m and V max were calculated for TK under the optimized experimental conditions. In addition, the inhibition effectiveness of two model inhibitors, Staurosporine and SU6656 were evaluated. The results indicated that the screening platform based on electrophoresis was suitable for TK analysis and laid a foundation for the HTS of TK inhibitors. [Copyright &y& Elsevier]

A water-soluble negative sulfonic propyl ether β-CD polymer (SPE-β-CDP) to be used as chiral selector in capillary electrophoresis (CE) was polymerized. The sulfonic substitution degree of each β-CD in SPE-β-CDP was statistically homogenized. The only one negative peak in electrophoretogram with indirect ultraviolate method proved its uniformity of electrophoretic behavior. There were 7.12 sulfonic substitution in β-CD unit and 164 μmole β-CD units in each gram of SPE-β-CDP, which corresponded a molecular weight of 7000 or more. Compared with monomer, SPE-β-CDP was lower effect on electrical current of CE, indicating a high concentration of SPE-β-CDP could be added. Its separation ability was verified by 12 chiral drugs. SPE-β-CDP also showed advantages of good water solubility, easy preparation and recovery to reduce the overall cost. However, five of 12 chiral drugs were hardly to be fully separated which was normal for any kind of chiral selector. A newly adjustable gravity mediated capillary electrophoresis (AGM-CE) technology was proposed and combined with SPE-β-CDP to enhance the chiral separation efficiencies of propranolol, salbutamol, omeprazole, ofloxacin and phenoxybenzamine which were markedly improved to 3.02, 1.17, 7.63, 4.14, and 2.81, respectively. Furthermore, its gradient mode (AGMg-CE) was also used to improve resolution through utilizing the zero mobility point, at which the effective apparent mobility of one racemate was zero. Resolutions of five chiral drugs were significantly improved, especially resolution of carvedilol changed from 0.43 to 1.0. These indicated SPE-β-CDP as chiral selector, AGM-CE and AGMg-CE as new CE technologies had a great potential in chiral separation. [Display omitted] • A adjustable gravity mediated CE (AGM-CE) was proposed and verified. • Gravity effect could be precisely controlled by simply tilting angle of capillary. • A gradient CE was proposed and applied in chiral separation. • The sulfonic substitution degree of polymer was statistically homogenized. • The chiral selector has high chiral separation ability and was recycle. [ABSTRACT FROM AUTHOR]

A smart design of transition metal nanocatalyst can efficiently improve its catalytic activity and use value. In this work, platinum (Pt) was successfully anchored to silica gel with phenylboronic acid group first time and formed nanoparticles (0.5–5 nm) which afforded a highly active supported catalyst for the hydrosilylation of olefins. The turnover number and regioselectivity in the reaction of methyldichlorosilane and 1-octene using 0.00000004 mol of the immobilized Pt catalyst approached 1,321,317 and 99% over 12 h. The corresponding hydrosilylation products in excellent yields and regioselectivities were obtained when we employed a broad range of terminal aliphatic olefins and methyldichlorosilane as substrates. The fully immobilized Pt nanoparticles could be recycled at least 10 runs without any obvious loss in catalytic activity. [ABSTRACT FROM AUTHOR]

Reduction in the sample complexity enables more thorough intact protein analysis using MS-based proteomics. A capillary electrophoresis method, namely the velocity gap mode of capillary electrophoresis (VGCE), is proposed to separate protein mixtures with high resolution. Although the separation mechanism of VGCE is also based on the difference of the mass-to-charge ratios of the proteins, it fractionates the sample zone into small pieces of subunits. In this way, the resolution can be dramatically improved due to less longitudinal dispersion of the sample. The effect of the new approach is evaluated by separation of three groups of reference protein mixtures, i.e. a mixture of lysozyme and BSA; a mixture of lysozyme, β-lactoglobulin, and ribonuclease A; and a mixture of cytochrome C, lysozyme, BSA, β-lactoglobulin, ribonuclease A, conalbumin, carbonic anhydrase, and hemoglobin. The results indicate that the new approach shows great potential to couple with MS for top-down analysis of complex mixtures. [ABSTRACT FROM AUTHOR]

Capillary electrophoresis (CE) has been applied to determine the percentage of enantiomeric excess (ee%) of chiral compounds. In such assays, the quality of chiral selectors (CSs) plays vital roles in resolving the enantiomers for accurate determination of the ee%. Selecting an efficient CS is usually by trial and error, and is, if ever possible, time-consuming and costly. Here we propose a new approach by using the velocity gap mode of CE (VGCE) method, to simplify the method development process for ee% determination. With VGCE, it is still possible to measure ee% even when the CS has a weak resolving power. This is especially important at the extreme cases where one of the enantiomers is significantly higher than the other one. The key point of VGCE in this case is to fractionate the small part of the mixture containing both enantiomers from the major component of the enantiomer, which is already enantiopure. Baseline separations can be achieved between the two enantiomers for the small mixture due to less longitudinal dispersion, making it possible to determine the ee%. The feasibility of this VGCE approach was confirmed by the ee% measurements of amlodipine and ofloxacin, respectively. And the practical application of VGCE was tested by analyzing levamlodipine besylate tablet. [ABSTRACT FROM AUTHOR]

In this paper, an active, recyclable and stable magnetic palladium nanocatalyst (Fe 3 O 4 @SiO 2 -DTPA-Pd) was reported which could catalyze Suzuki coupling reaction. The Pd was stably hold by the three nitrogen atoms at the inside and four acetic acid groups at the outside of the chelating ligand ethylenediaminepentaacetic acid (DTPA) on the surface of core-shell magnetic Fe 3 O 4 @SiO 2 microspheres. The prepared Fe 3 O 4 @SiO 2 -DTPA-Pd was systematically characterized. Results showed its size was 10–20 nm with 0.535 mmol/g Pd2+/Pd0. It could monodisperse in liquid, was thermally stable and good magnetic response from the high saturation magnetization value at 35.9 emu/g. Its activity was evaluated through Suzuki coupling reactions between 14 types of aryl halides and phenylboronic acid under optimized reaction conditions. It exhibited excellent activities and good applicability to substituted iodobenzene and bromobenzene. The yield of 4-bromobenzonitrile was up to 97.93%. Furthermore, the Fe 3 O 4 @SiO 2 -DTPA-Pd catalyst was stable, reusable and conveniently recovered by applying an external magnetic field. After 20 consecutive reaction cycles, the yield was still above 85%, which showed a superior heterogeneous advantage over other catalysts reported in the literature and had the huge potential in actual application of industry. [Display omitted] • Chelating agents DTPA modified Fe 3 O 4 @SiO 2 anchored Pd nanocatalyst was developed and used for catalyzing Suzuki coupling reactions. • The activity of the immobilized nanocatalyst (0.17 mol% Pd) was better than homogeneous Pd(OAc) 2 (1.00 mol% Pd). • Facile recycle for 20 consecutive runs with yield still over 85% exhibited a good reusability of Fe 3 O 4 @SiO 2 -DTPA-Pd. [ABSTRACT FROM AUTHOR]

A novel 96-well liquid-liquid microextraction system combined with modern HPLC was developed and used for the simultaneous analysis of 96 biological samples. The system made use of hollow fibers, a 96-well plate, and a plastic base with a center hole and a side hole. One end of the hollow fiber was sealed, while the other end was attached to one of the holes positioned at the center for the plastic base. The needle was inserted into the liquid from inside or outside of the hollow fiber through the center or the side holes, respectively. The system was tested with plasma samples containing three compounds, acidic indomethacin, neutral dexamethasone, and basic propafenone. Some parameters, such as the kind and dimension of hollow fiber, pH and salt concentration of the donor phase, the selection of organic solvent for the acceptor phase, and the extraction time were investigated. Under the optimization conditions, the Log D and drug concentration of indomethacin, dexamethasone, and propafenone in plasma and urine samples were analyzed. Then, the methodology was validated. The results demonstrated that ng/mL levels could be exactly and rapidly analyzed by our system, which was equipped with an auto-injection sampler, making sample analysis more convenient. [ABSTRACT FROM AUTHOR]

Nanoparticles modified with aminopropyl group were prepared by a single-step electrospray process and used as a pseudo-stationary phase for the separation of anions, (e.g., organic acids in capillary electrophoresis). The nanoparticles form a uniform suspension which is stable in a buffer without adding other stabilizer. The amino containing nanoparticles have significant impact on the electro-osmotic flow (EOF) of the system, which, in turn, affects the overall separation of other analytes in this system. For example, five organic acids, which were not all separable in zone electrophoresis, were fully resolved in the presence of the nanoparticles in a wide range of pH. Various experimental parameters that are related to the electrophoresis including EOF, pH, organic modifier, voltage, concentration of the running buffer and concentration of the nanoparticles have been investigated. The repeatability of the migration time and peak area in this system are better than 5% for both inter- and intra-day evaluation. [ABSTRACT FROM AUTHOR]

Multiplexed (96-lane) capillary electrophoresis with laser-induced fluorescence (MCE–LIF) detection was used for the rapid analysis of extracellular signal-regulated protein kinase (ERK) levels from in vitro cell extracts. The levels of ERK enzyme in cell extracts were determined by monitoring the conversion of a fluorescent-labeled peptide substrate to a phosphorylated fluorescent-labeled peptide product using MCE–LIF. The incorporation of a fluorescent internal standard was found to improve the precision of the analysis. The enzyme assay conditions including substrate concentration, reaction time and enzyme linear range were rapidly optimized using the MCE–LIF approach for both direct and immunoprecipitation-based ERK assays. The levels of ERK from in vitro cell extracts stimulated with angiopoietin 1 (Ang1*) were determined using the MCE–LIF approach. The advantages of MCE–LIF for developing and applying enzyme assays, as well as the figures of merit for the direct and immunoprecipitation ERK assays, are discussed. [Copyright &y& Elsevier]

The application of microchip capillary electrophoresis (CE) to the assay of extracellular signal-regulated protein kinase (ERK) is presented. In this assay, ERK catalyzes the transfer of γ-phosphate from adenosine 5′-triphosphate to the threonine residue of a fluorescently labeled nonapeptide (APRTPGGRR), and the phosphorylated and nonphosphorylated peptides were detected by fluorescence. The phosphorylated and nonphosphorylated peptides and the internal standard were separated within 20 s, and the increase in magnitude of the phosphorylated peptide peak was monitored to assess ERK activity. ERK reactions were prepared off-chip and analyzed on a single-lane glass microchip fabricated by standard methods. It was demonstrated that microchip CE could be used to measure endogenous amounts of ERK by spiking known concentrations of recombinant ERK2 into the lysates of serum-starved human umbilical vein endothelial cells (HUVEC) and recovering between 90 and 100% for all samples. Endogenous ERK activity was determined by microchip where HUVEC were stimulated with 500 pM vascular endothelial growth factor (VEGF) at different times before cell lysis. The results showed a transient VEGF-mediated ERK activation that peaked at 10 min, which was consistent with previous reports using conventional techniques. The microchip assay provided a rapid, accurate, and precise alternative to conventional methods of determining endogenous ERK activity. [Copyright &y& Elsevier]