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In the microbiome, probiotics modulate oral diseases. In this study, Streptococcus strain C17T was isolated from the oropharynx of a 5-year-old healthy child, and its potential probiotic properties were analysed using human bronchial epithelial cells (16-HBE) used as an in vitro oropharyngeal mucosal model. The results demonstrated that the C17T strain showed tolerance to moderate pH ranges of 4–5 and 0·5–1% bile. However, it was more tolerant to 0·5% bile than 1% bile. It also demonstrated an ability to accommodate maladaptive oropharyngeal conditions (i.e. tolerating lysozyme at 200 μg ml−1). It was also resistant to hydrogen peroxide at 0·8 mM. In addition, we found out that the strain possesses inhibitory activities against various common pathogenic bacteria. Furthermore, C17T was not cytotoxic to 16-HBE cells at different multiplicities of infection. Scanning electron microscopy disclosed that C17T adhesion to 16-HBE cells. Competition, exclusion and displacement assays showed that it had good anti-adhesive effect against S. aureus. The present study revealed that Streptococcus strain C17T is a potentially efficacious oropharyngeal probiotic.

In the microbiome, probiotics modulate oral diseases. In this study, Streptococcus strain C17

In the current study, two Cu(II)-based coordination polymers with the molecular formula of {[Cu3(μ3-cpia)2(phen)3]·10H2O}n (1, H3cpia = 4-(6-carboxy-pyridin-3-yl)-isophthalic acid, phen = 1,10-phen...

In this paper, an improved trappy alpha-beta (simply) algorithm, namedis proposed.is an extended version ofby introducing the two-ply iterative deepening (TPID). By applying TPID, can search more deeply and therefore find the traps hidden more deep plies in game-tree. The experimental results reflect thatindeed obtains the better chess playing performance than.

The analysis of the residual life of high temperature low cycle fatigue of 30CrMnSiA steel plays important roles in improving security and avoiding accidents. In this paper, the RBF neural network method is used to predict the residual life of high temperature low cycle fatigue of 30CrMnSiA steel base on data from the thermo-mechanical fatigue test. The feasibility of the method is proved by a practice example, and the learning results are in good agreement with the experimental data.

The toughness of cement-based materials, related to the performance such as durability and strength, attracts much more attention nowadays. In this paper, the curve of bending load-deflection was adopted to research the influencesof polymers (dispersibility emulsion powder, PVA and styrene butadiene latex) and fibers (PVAF, steel fiber,basalt fiber) on basic mechanical properties and toughness. The results show that the PVAF pre-incorporate in cement motor could improve the toughness of cement motor. In addition, an environmental scanning electron microscopic analysis (ESEM) and hardness were adopted to analyze the microscope morphology of polymer films inside of hardened cementitious composites.

The quality improving of locomotive maintenance service is studied based on Six Sigma. The maintenance service progress has been optimized by DMAIC mode. First of all, the quality problems have been proposed. Then, the factors which impact on quality of after-sales service are found by QC tools. Secondly, the main problems in the progress would be controlled conveniently. Finally, the improved maintenance services process is developed correspondingly. In order to ensure the quality of the maintenance in the delivery period, some measures are taken such as adjusting the allocation time in the repair process, developing a strict examination system. The result shows that the maintenance efficiency and customer satisfaction has been improved significantly.

Energy saving is the best way to energy exploitation and environment protection, especially in the case of the low carbon economy. In this paper, a hybrid approach combining particle swarm optimization (PSO) and genetic algorithms (GA) is developed for optimization design of marine waste heat recovery system. Based on exergy analysis method, the waste heat utilization degree of the marine waste heat recovery system is discussed. Considering the superheated steam temperature, evaporation temperature, economizer inlet temperature, water supply temperature and condensation temperature, a mathematical model is constructed in which the exergy efficiency of marine waste heat recovery system is taken as the objective function for optimization design of marine waste heat recovery system. The mutation operator of GA is introduced to the PSO to solve the mathematical model of marine waste heat recovery system for the diversity of particles. The effectiveness of the approach is illustrated by an optimization example.

Context-aware applications can sense and explore the context of production processes to provide proper and useful services to managers in workshop. The monitoring levels are partitioned and designed in this article. And the multi-level ontology model of materials is found which is expressed in the Web Ontology Language (i.e. OWL) as a ontology collection of describing places ages and events and associated properties among them. Within an intelligent monitoring system, dynamic adjusting algorithm of linking local coordination with global coordination for lacking parts is brought forward and dynamic coordination structure is put up based on event-driven architecture. Context-aware reactive behaviors can be expressed by using rules written in OWL Language, especially developed for context-aware applications.

Three dimension temperature fields and stress-strain fields of the 6110-type diesel engine piston were analyzed by using the program ANSYS. The simulating tests of crack propagation under high temperature low cycle fatigue were carried out with pre-crack notched specimens. According to the theory of high temperature strength and fracture, the equivalent J-integral range ΔJf were induced to predict the surplus fatigue life of diesel engine piston. It was concluded that this approach is suitable and provides valuable reference for designers of diesel engine.

By the investigation of the 400°C low cycle fatigue behavior of 4 kinds of notched cylindrical specimens made 2.25Cr-1Mo stee1. The high temperature low cycle fatigue life was evaluated by the criterons of equivalent range. The results show that equivalent criterion for evaluating high temperature low cycle fatigue life has its experimental and theoretical foundation, but further investigation is still needed．

Aiming at the complexity of mechanical devices and the polytrope of operating conditions for marine propulsion plant, the modeling and simulation of propeller and dull system are investigated based on MATLAB/Simulink. The simulation model of propeller and dull system is constructed in which the Chebyshev fit expression across four quadrants is given for the propeller. So it becomes practical to express static and dynamic properties of propeller and dull system. A luxury cruises fitted on two engines and two fixed pitch propellers is considered to perform simulation tests. The actual navigation conditions of marine propulsion plant, including starting, parking and reversing etc, are taken into account. The simulation results analysis illustrates the correctness and validity of modeling and simulation for propeller and dull system. Thus provides a new method for the optimization and design of marine propulsion plant.

The crack propagation law of 2.25Cr-1Mo steel with notched cylinder was researched under high temperature low cycle fatigue. The crack propagation life was viewed by fatigue experiment and the equivalent stress-strain on the crack tip was calculated by the ANSYS. The equivalent J-integral range which was computed by equivalent elastic and plastic strain ranges were employed to denote the fatigue crack propagation rate. The results showed that crack propagation law of this material under complex stress state can be characterized by equivalent J-integral ranges and the relation between da/dN and ΔJf is not influenced by the type of notch and the load strain range.

In order to solve the fault diagnosis problem of diesel engine, Elman neural network (ENN) was applied to build a fault diagnosis model of diesel engine. The training algorithm is in introduced and at the same time, the process of diesel engine fault diagnosis is also expatiated. The diagnosis results indicate the reliability. So a contingent fault of diesel engine can be identified effectively.

The low cycle fatigue behavior was experimentally studied with the 3-dimension notched LD8 aluminum alloy specimens at 300°C. The 3- dimension stress-strain responses of specimens were calculated by means of the program ADINA. The multiaxial fatigue life prediction was carried out according to von Mises’s equivalent theory. The results from the prediction showed that the equivalent strain range can be served as the valid mechanics for predicting multiaxial high temperature and low cyclic fatigue life.

For the cooling system of plastic injection mold affects significantly the productivity and quality of the finial products, the cooling system design is of great importance. In this paper, considering the location, size, number of cooling channels and mold material properties, the optimization design of heterogeneous injection mold cooling system is discussed to achieve simultaneously fast and uniform cooling. A mathematical model is constructed in which the part temperature distribution uniformity and production efficiency are taken as the objective function for cooling system design of heterogeneous injection mold. For the reason of the non-linearity of design problem and the coupling of geometric with material variable, the design problem is divided into two sub-problems and the combining algorithm is used in such way that, the geometric optimization sub-problem is solved by Hooke-Jeeves method and the material optimization sub-problem is solved by particle swarm optimization (PSO) method. The effectiveness of the approach is illustrated by a design example of injection mold cooling system.

Matching performance of ship engine and propeller has a significant impact on marine propulsion efficiency. In this paper, a hybrid approach combining particle swarm optimization (PSO) and genetic algorithms (GA) is developed for matching optimization of ship engine and propeller. Based on ship theory, the matching performance of ship engine and propeller is analyzed. Considering the diameter, angular speed, picth ratio and disk ratio of propeller, a mathematical model is constructed in which the open-water propeller efficiency is taken as the objective function for matching optimization of ship engine and propeller. Integrating PSO with GA is presented to solve it, in which the mutation operator of GA is introduced to the PSO for the diversity of particles. The effectiveness of the approach is illustrated by a matching optimization example of ship engine and propeller.

The structure, characteristics and principles of BP neural network model are described in this paper. First, three impact factors of the dissolved oxygen are selected as the sample input of network, and then the parameters of BP neural network are selected, such as network structure, learning algorithm, output layer transfer function, learning rate and so on. Finally, the BP neural network model is established and trained, in order to approach compensate the effects of improves non-linearity. The simulation results show that BP neural network is practical and dependable in the field of dissolved oxygen modeling and has nice applied prospect.

A new topology optimization model with holes’ geometric constraints for continuum structure is presented. It is solved by an evolutionary optimization method based on interval relaxation, in which the problem is divided into two subproblems of topology optimization process and size/shape optimization process. The optimal topology of structure can be found gradually by introducing interval relaxation factor to adjust holes’ size constraints, delete noneffective holes and by generating new holes based on the sensitivity analysis of objective function. Interior penalty-function method is employed as an optimization technique for the size/shape optimization of the structure corresponding to the topology. When the holes’ size bounds are the actual values, the optimal solution is the smallest objective function structure in the various topologies. Thus realizes the holes’ geometric size and location design, topology design and layout design together. The optimization results of example shows the method proposed is of good effectiveness and engineering applicability.

In this paper, the tests of the thermo-mechanical fatigue of the material, cut from a coke tower were performed. The tower made of 20G has been in service for 20 years. Then, we calculated the temperature and stress-strain. The results showed that the tower can be in service for another four years．

The in-phase and out-of-phase thermal fatigue of aluminum alloy were experimentally studied. The fatigue life was evaluated analytically by using the elastic-plastic fracture mechanics method (mainly J integral). The results of experiments and calculations showed that the life of out-of-phase fatigue was longer than that of in-phase fatigue within the same strain range. This is the same as the results of other materials such as medium and low carbon steel. On the other hand, the predicted life was consistent with experimental results. This suggests that J integral as a mechanics parameter for characterizing the thermal fatigue strength of aluminum alloy and the calculation method developed here is efficient. A parameter ΔW was proposed from energy aspect to characterize the capacity of crack propagation. The in-phase thermal fatigue life was the same as the out-of-phase thermal fatigue life for identical ΔW values.

This paper addresses on quality improvement of expanded food. The five stages of six sigma implementation process are applied to define the problem and set a target for improvement. Then possible causes to the quality problems in expanded food are investigated, and the capabilities of the measuring system are evaluated and improved. Based on the results, the experiment scheme is conducted to find and optimize the key factors. Improvement result shows that sigma level of production process increased to 4.6, which is beyond the expectation and can save the cost.

The high temperature & low cycle fatigue were experimentally studied with the 3-dimension notched 2.25Cr—1Mo steel specimens. The 3-dimension stress-strain responses of specimens were calculated by means of the program ADINA. The prediction method of 3-dimension fatigue life was discussed according to the theory of high temperature strength. The results of experiments and calculations showed that the equivalent strain range can be served as the valid mechanics for predicting fatigue life.

Novel 1-(2-aminopyrazin-3-yl)methyl-2-thioureas are described as inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2). These compounds demonstrate potent in vitro activity against the enzyme with IC(50) values as low as 15 nM, and suppress expression of TNFalpha in THP-1 cells and in vivo in an acute inflammation model in mice. The synthesis, structure-activity relationship (SAR), and biological evaluation of these compounds are discussed.

In this paper, a dynamic competitive swarm optimizer (DCSO) based on population entropy is proposed. The new algorithm is derived from the competitive swarm optimizer (CSO). The new algorithm uses population entropy to make a quantitative description about the diversity of population, and to divide the population into two sub-groups dynamically. During the early stage of the execution process, to speed up convergence of the algorithm, the sub-group with better fitness will have a small size, and worse large sub-group will learn from small one. During the late stage of the execution process, to keep the diversity of the population, the sub-group with better fitness will have a large size, and small worse sub-group will learn from large group. The proposed DCSO is evaluated on CEC'08 benchmark functions on large scale global optimization. The simulation results of the example indicate that the new algorithm has better and faster convergence speed than CSO.

The particle image velocimetry (PIV) technique was used to study the fluid flow phenomena that occurred during continuous casting, using a water model with dimensions of 1 840 mm × 280 mm. Two types of solidified shells, i. e., the smooth type and the coarse type, were used to characterize the dendrite in order to simulate different liquid-solid interfacial conditions. The influence of the nozzle angle and the immersion depth of nozzle, as well as the casting speed on the flow behavior was investigated quantitatively. The results were as follows: (1) There are two large recirculations above and below the fluid jet in the mold, respectively, under the smooth interface condition. However, in the case of the dendrite solidified shell, it was found that the flow velocity of the fluid decreased and more smaller vortices appeared in the upper region of the mold. (2) The angle and the immersion depth of nozzle are two important factors affecting the flow pattern, and they are also capable of bringing about the change in the flow direction. (3) The higher the casting speed, the higher are the jet stream and the impacting point on the narrow face. However, the high casting speed causes serious fluctuation of the meniscus, and correspondingly leads to various defects.

To study the effect of 1,4-naphthoquinone aged black carbon (BC/1,4-NQ) on reactive oxygen species and DNA strand breaks in human bronchial epithelial cells (16HBE).In the study, 16HBE cells were exposed to BC/1,4-NQ, BC and 1,4-NQ at the concentrations of BC/1,4-NQ (10.0/0.2, 20.0/0.4, 40.0/0.8, 80.0/1.6, 160.0/3.2 mg/L), BC (10.0, 20.0, 40.0, 80.0, 160.0 mg/L), 1,4-NQ (0.2, 0.4, 0.8, 1.6, 3.2 mg/L) for 24, 48, and 72 h, respectively. Cytotoxicity was detected by cell count kit 8 (CCK-8) at the end point. Then the 16HBE cells were exposed to BC/1,4-NQ (20.0/0.4, 40.0/0.8, 80.0/1.6 mg/L), BC (20.0, 40.0, 80.0 mg/L), 1,4-NQ (0.4, 0.8, 1.6 mg/L) for 24 h. The reactive oxygen species (ROS) generation was determined via flow cytometry with DCFH-DA probe. Single cell gel electrophoresis (SCGE) assay was performed to evaluate genotoxicity by Olive tail moment (OTM) value.Except for the concentration of 10.0/0.2 mg/L within the exposure time 24 h, the cell viabilities of BC/1,4-NQ were significantly lower than the control (P0.05) within the exposure time 24-72 h, showing a dose-dependent cytotoxicity. Especially, BC/1,4-NQ showed greater cytotoxicity than BC single exposure, lower than 1,4-NQ at the concentration of BC/1,4-NQ≥80.0/1.6 mg/L. BC/1,4-NQ also showed greater ROS generation and OTM value than the control within the exposure time 24 h at each concentration (P0.05). Especially, the ROS generation and OTM value of BC/1,4-NQ were greater than BC single exposure, lower than 1,4-NQ at the concentration of 80.0/1.6 mg/L (P0.05).BC/1,4-NQ can induce intracellular ROS generation, cytotoxicity and genotoxicity in 16HBE cells. And at high concentration, the intracellular ROS level, cytotoxicity and genotoxicity induced by BC/1,4-NQ were greater than those by BC single exposure, but lower than those by 1,4-NQ.

Several series of novel non-thiourea-containing aminopyrazine derivatives were designed based on the MK-2 inhibitors 1-(2-aminopyrazin-3-yl)methyl-2-thioureas. These compounds were synthesized and evaluated for their inhibitory activity against MK-2 enzyme in vitro. Compounds with low micromolar to sub-micromolar IC50 values were identified, and several compounds were also found to be active in suppressing the lipopolysaccharide (LPS)-stimulated TNFα production in THP-1 cells with minimum shift compared to their enzyme activity.

Quinolinones and naphthyridinones with C7 N-t-butyl piperidine substituents were found to be potent p38 MAP kinase inhibitors. These compounds significantly suppress TNF-alpha release in both cellular and LPS-stimulated whole blood assays. They also displayed excellent PK profiles across three animal species. Quinolinone at 10 mpk showed comparable oral efficacy to that of dexamethasone at 1 mpk in a murine collagen-induced arthritis model.

With the rapid development of computer technology and Internet, all kinds of distant educations based on Internet have been coming out constantly. To reduce the cost of exam and alleviate the burden of teachers, the crew examination system was designed and implemented. This system is an online examination system which integrates computer network technology, database technology and ASP technology. The B/S model was adopted in this system and using ASP visit ODBC accesses database. This system includes two functions: examination system and management system. Test library management, random questions, online testing, automatic scoring, scores query and other functions were achieved. Crew examination system realizes the change from traditional test’s way to the way based on Internet.

To make an assessment on the genotoxicity caused by black carbon (BC) and ozonized black carbon (O₃-BC).In this study, 74 healthy male ICR mice [weighed (28 ± 1.5) g] were randomly divided into 7 groups, including one phosphate buffer solution (PBS) control group and six particles exposed groups by intratracheal instillation with either BC or O₃-BC at the doses of 50, 100, 200 μg/mouse, respectively. There were 12 mice in the groups of 200 μg/mouse and 10 mice in others. The mice were sacrificed 24 h after four intratrachealinstillations. The activities of catalase (CAT) in serum and the levels of malondialdehyde (MDA) in lung tissue homogenate were measured. As the DNA damage mark, 8-hydroxyguanosine (8-OHdG) in urine and serum were quantified with ELISA method. Micronucleus test was used for potential genotoxicity of BC and O₃-BC. Hematoxylin and eosin staining was used to stain lung paraffin section.The mice were in good condition during instillation, and the liver coefficient of the test groups was significantly lower than that of the control group (P0.05). The activities of CAT in serum significantly increased in the 100 μg/mouse and 200 μg/mouse groups after being exposed to these two kinds of particles. The micronucleus rate in allthe BC and O₃-BC exposed groups increased (P0.05), but there was no statistically significant difference among the groups in the levels of 8-OHdG in serum and urine and MDA in lung tissue homogenate. Inflammatory response was found in the lung tissue under the microscope after exposure to BC and O₃-BC.Intratracheal instillation of BC and O₃-BC induced increasing of oxidative stress and genetic damage in mice. But there was no significant difference between these two particles in toxicity. Whether the genotoxicity of O₃-BC is higher than that of BC or not is uncertain. Further research is needed.

Objective: To assess the effects of perioperative comprehensive supportive care interventions on outcome of Chinese esophageal cancer patients in a prospective study. Methods: 60 patients with primary esophageal carcinoma were randomized into an intervention group (IG, n=31) and a control group (CG, n=29). The Chinese version of symptom checklist-90 (SCL-90) was adopted to assess their psychological status. The interventions, including health education, psychological support, stress management, coping strategies and behavior training, were carried out in 3 phases (preoperative, postoperative Ⅰ and postoperative Ⅱ), and psychological effects were thereafter evaluated accordingly before surgery, and 1 week, 4 weeks and 24 weeks post-surgery. Medical costs were estimated at discharge. Survival of patients was estimated each year post-surgery. General health status and satisfaction-with-hospital were surveyed by a follow-up questionnaire 4 years post-surgery. Results: All the subjects demonstrated higher scores in the preoperative phase than the normal range of Chinese population concerning 7 psychological domains including somatization, obsessive-compulsive, depression, anxiety, hostility, phobic anxiety and paranoid ideation. Although no significant difference was observed between the two groups at admission, the scores of IG, which tended to decrease at a faster rate, were generally lower than those of CG at weeks 1, 4 and 24 post-surgery. The length of hospital stay and medical costs of IG were significantly less than those of CG and satisfaction-with-hospital was better. However, there was no significant difference in 4-year survival or health status between two groups. Conclusions: Appropriate perioperative comprehensive supportive care interventions help to improve the psychological state of Chinese patients with esophageal carcinoma, to reduce health care costs and to promote satisfaction of patients and their families with hospital.

Summary Background Although unsupported upper extremity exercise (UUEE) is recommended in the guidelines for pulmonary rehabilitation (PR), it is controversial whether UUEE improves dyspnea in patients with COPD. The present study conducted a meta-analysis of randomized controlled trials to clarify whether UUEE could improve dyspnea in COPD patients. Methods A computerized search through PubMed and Embase (up to Mar 2012) was performed to obtain sample studies. Methodological quality was assessed using the PEDro scale. Weighted mean differences (WMDs), and 95% confidence intervals (CIs) were calculated and heterogeneity was assessed with the I 2 test. The overall effect sizes were compared with the minimum clinically important difference (MCID). Results 240 patients from 7 studies were included in this meta-analysis. The mean PEDro score was 7.0 (SD = 1.7). The results indicated UUEE relieved dyspnea and arm fatigue during activities of daily living (ADL) (WMD = −0.58, −0.55 scores; 95% CI = −1.13 to −0.02, −1.08 to −0.01), however, the overall treatment effects were lower than the MCID of 1 unit for the Borg scale. There was no statistical significance for dyspnea and arm fatigue during intervention (WMD = −0.34, 0.24 scores; 95% CI = −0.78 to 0.09, −0.33 to 0.81). Conclusions UUEE can relieve dyspnea and arm fatigue in patients with COPD during ADL and should be included in the PR program, however, there is currently a lack of clinical evidence to support UUEE relieving dyspnea and arm fatigue. Further study is urgent to investigate these effects of UUEE.

To investigate the protective effect of polyenylphosphatidyl choline (PPC) on liver in rat with sepsis.A total of 50 healthy male SD rats were randomly divided into four groups according to random number table: normal control group (NS group, n=10), sepsis model group (LPS group, n=15), PPC control group (PPC group, n=10) and PPC protection group (P+L group, n=15). A single dose of lipopolysaccharide (LPS) 6 mg/kg was injected to the peritoneal cavity to reproduce the sepsis model, while in NS group and PPC group, same volume of normal saline was used. PPC 10 ml/kg (232.5 mg/kg) was injected 24 hours and 6 hours before LPS via the tail vein in PPC group and P+L group, while in NS group and LPS group 5% glucose solution in the same volume was given. Behavioral changes and mortality rate of rats were recorded, and all survived rats were sacrificed 24 hours after LPS injection. Venous blood was taken for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) determination. Ratios of liver weight/body weight (L/Wb) and wet weight/dry weight of liver (W/D) were calculated. Histopathology changes in liver tissue were observed with hematoxylin eosin (HE) stain, and intercellular adhesion molecule-1 (ICAM-1) expression with instant two-step immunohistochemistry.Twenty-four hours after LPS injection (LPS group), the rats became lethargic, with less activity and drinking, while rats in P+L group, showed much better mental status, water intake and activity than LPS group. The mortality rate of P+L group was significantly lower than that of LPS group [6.7% (1/15) vs. 46.7% (7/15),P0.05]. Compared with LPS group, rats in P+L group had lower levels of plasma ALT and AST, L/Wb, W/D , and liver ICAM-1 positive expression ratio [ALT (U/L): 157.71 ± 32.63 vs. 225.63 ± 43.47; AST (U/L): 53.21 ± 13.85 vs. 85.25 ± 18.91; L/Wb: (4.09 ± 0.28)% vs. (4.50 ± 0.25)%; W/D : 3.52 ± 0.27 vs. 3.84 ± 0.18; ICAM-1 positive expression ratio: 35.7% (5/14) vs. 87.5 (7/8),P0.05 or P0.01]. All parameters were normal in NS group and PPC group, and no statistical significance was observed between them (all P0.05). LPS-injection led to severe inflammatory reaction in hepatic tissue , including obvious edema of hepatic parenchymal cells, exudation and aggregation of inflammatory cells, while in P+L group, these morphological changes were milder than that in LPS group but more obvious than that of NS group.Pre-treatment of rats with PPC alleviates progressive inflammatory disturbances of hepatic tissue caused by endotoxin injection, and it attenuates liver edema and ICAM-1 expression, protects liver function and decreases mortality rate in rats with sepsis.

To meet the strict requirement of International Marine Organization (IMO) Tier III regarding emission from marine diesel engines, many researchers have been conducted at marine vessels to reduce exhaust emissions from large diesel engines. This paper introduces many methods to control emission of NOx. Based on some particular types of engines, the emphasis is put upon the technologies currently employed onboard to reduce the NOx emission, especially those technologies utilized overseas in this field with more potential. Finally, trend of development in this field is proposed as well.

We have developed assays for the binding of nucleotide and protein substrates to p38alpha protein kinase based on time-resolved Forster resonance energy transfer. p38alpha was biotinylated by addition of a sequence that targets biotin to a single lysine when coexpressed with biotin ligase in Escherichia coli, allowing formation of a complex between a streptavidin "LANCE" europium chelate conjugate and p38alpha. When this reagent was combined with M39AF, a p38 inhibitor containing a fluorescent moiety whose excitation wavelengths match the emission wavelengths of the europium chelate, a change in ratio of light emitted at 665 nm/615 nm is detected. Less than 100pM complex was detected with a signal/background ratio of30-fold. The complex exhibits slow, tight binding kinetics where the apparent K(d) decreases with a relaxation time of 21 min at 125 pM biotin-p38alpha. Preincubating inhibitors or ATP with biotin-p38alpha and adding M39AF as a competitor yielded IC(50)s consistent with those measured by enzyme assay for the activated form of biotin-p38alpha. The same technique was also used to measure affinity of inhibitors for the unphosphorylated and catalytically inactive form of biotin-p38alpha. To measure affinity of p38alpha for its protein substrate MK2, we incubated biotin-p38alpha with a glutathione S-transferase MK2 fusion protein. Detection of the complex after incubation with streptavidin-allophycocyanin and a LANCE-conjugated anti-GST allowed measurement of affinity of MK2 for biotin-p38alpha and detection of 0.5 nM p38alpha.MK2 complex with signal/background ratio5-fold. Competition with unbiotinylated p38alpha yielded an IC(50) value of 5 nM. Activation of either p38alpha or MK2 had no effect on the measured K(d). M39AF was found to bind in a ternary complex with p38alpha.MK2 with lower affinity than that observed in the binary complex with p38alpha alone.

Non-adherence with medication remains a major correctable cause for poor outcome in schizophrenia. We describe a surgically implantable preparation of haloperidol with the aim that patients will have superior outcomes with improved medication adherence from implants. In contrast to depot formulations, implantable pellets could last many months, providing symptomatic improvement for periods of time never before possible. Additionally, in the event of unacceptable side effects, implants could be removed, offering a degree of reversibility not available with depot formulations. A surgically-implantable formulation of haloperidol has been created using biodegradable polymers. Implants have been characterized for in-vitro kinetics, as well as in-vivo bioactivity in rodents. Haloperidol implants demonstrate steady release of drug for 5 months. Animals treated with haloperidol implants display increased striatal D2 receptor expression as well as increased apomorphine stimulated locomotion. Surgically-implantable formulations are a viable approach to provide long-term delivery of antipsychotic medications to patients with psychotic disorders.

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.

We report here our preliminary investigations on the mechanism of alpha-TTP-mediated ligand transfer as assessed using fluorescence resonance energy transfer (FRET) assays. These assays monitor the movement of the model alpha-tocopherol fluorescent derivative ((R)-2,5,7,8-tetramethyl-chroman-2-[9-(7-nitro-benzo[1,2,5]oxadiazol-4-yl amino)-nonyl]-chroman-6-ol; NBD-Toc) from protein to acceptor vesicles containing the fluorescence quencher TRITC-PE. We have found that alpha-TTP utilizes a collisional mechanism of ligand transfer requiring direct protein-membrane contact, that rates of ligand transfer are greater to more highly curved lipid vesicles, and that such rates are insensitive to the presence of anionic phospholipids in the acceptor membrane. These results point to hydrophobic features of alpha-TTP dominating the binding energy between protein and membrane.