G protein coupled receptors -in silico drug discovery and design.

In silico drug discovery is a complex process requiring flexibility and ingenuity in method selection and a careful validation of work protocols. GPCR in silico drug discovery poses additional challenges due to the paucity of crystallographic data. This paper starts by reviewing selected GPCR in silico screening programs reported in the literature, including both structure-based and ligand-based approaches. Particular emphasis is given to library design, binding mode selection, process validation and compound selection for biological testing. Following literature review, we provide insights into in silico methodologies and process workflows used at EPIX to drive over 20 highly successful screening and lead optimization programs performed since 2001. Applications of the various methodologies discussed are demonstrated by examples from recent programs that have not yet been published.

Clowns for the prevention of preoperative anxiety in children: a randomized controlled trial.

OBJECTIVE: To determine if specially trained professional clowns allayed preoperative anxiety and resulted in a smooth anesthetic induction compared to the use of midazolam or no intervention. METHODS: This was a randomized, controlled, and blinded study conducted with children 3-8 years of age undergoing general anesthesia and elective outpatient surgery. Patients were assigned to one of three groups: Group 1 did not receive midazolam or clown presence; group 2 received 0.5 mg x kg(-1) oral midazolam 30 min before surgery up to a maximum of 15 mg; and group 3 had two specially trained clowns present upon arrival to the preoperative holding area and throughout operating room (OR) entrance and mask application for inhalation induction of anesthesia. The children were videotaped for later grading. RESULTS: The clown group had a statistically significant lower modified-Yale Preoperative Anxiety Scale score in the preoperative holding area compared to the control and midazolam group. The clowns' effect on anxiety reduction continued when the children entered the OR but was equal at this point to the midazolam group. Upon application of the anesthesia mask no significant differences were detected between the groups. CONCLUSIONS: This study found that the use of preoperative medically trained clowns for children undergoing surgery can significantly alleviate preoperative anxiety. However, clowns do not have any effect once the anesthesia mask is introduced.

Large renal angiomyolipomas: digital subtraction angiographic grading and presentation with bleeding.

AIM: To investigate whether a grading system of renal AMLs based on digital subtraction angiography (DSA) and computerized tomography (CT), could help to select patients for embolization. MATERIALS AND METHODS: Thirty patients with 35 renal angiomyolipomas (size range 4-20 cm, mean 9.9 cm) underwent both digital subtraction angiography (DSA) and computed tomography (CT). Based on the DSA appearance the tumours were graded into three grades: grade I, minimal vascularity; grade II, moderate vascularity; grade III, marked vascularity. RESULTS: There were seven grade I (mean 9.3 cm, range 4.5-20 cm), 18 grade II (mean 8.9 cm, range 5-18 cm) and 10 grade III tumours (mean 12.1 cm, range 4-20 cm). Five grade I tumours did not cause symptoms (71%) and two caused flank pain and haematuria, respectively (14.3% each). Nine of the grade II tumours were asymptomatic (50%), seven caused bleeding (39%) and two caused flank pain (11%). Four grade III tumours were asymptomatic (40%), five caused bleeding (50%) and one pain (10%). CONCLUSIONS: According to our criteria, large angiomyolipomas with minimal vascularity are less likely to bleed, and do not need prophylactic treatment. This needs to be confirmed in larger studies.

Ethanol sclerotherapy of peripheral venous malformations.

BACKGROUND: Venous malformations are congenital lesions that can cause pain, decreased range of movement, compression on adjacent structures, bleeding, consumptive coagulopathy and cosmetic deformity. Sclerotherapy alone or combined with surgical excision is the accepted treatment in symptomatic malformations after failed treatment attempts with tailored compression garments. OBJECTIVES: To report our experience with percutaneous sclerotherapy of peripheral venous malformations with ethanol 96%. PATIENTS AND METHODS: 41 sclerotherapy sessions were performed on 21 patients, aged 4-46 years, 15 females and 6 males. Fourteen patients were treated for painful extremity lesions, while five others with face and neck lesions and two with giant chest malformations had treatment for esthetic reasons. All patients had a pre-procedure magnetic resonance imaging (MRI) study. In all patients, 96% ethanol was used as the sclerosant by direct injection using general anesthesia. A minimum of 1-year clinical follow-up was performed. Follow-up imaging studies were performed if clinically indicated. RESULTS: 17 patients showed complete or partial symptomatic improvement after one to nine therapeutic sessions. Four patients with lower extremity lesions continue to suffer from pain and they are considered as a treatment failure. Complications were encountered in five patients, including acute pulmonary hypertension with cardiovascular collapse, pulmonary embolus, skin ulcers (two) and skin blisters. All patients fully recovered. CONCLUSION: Sclerotherapy with 96% ethanol for venous malformations was found to be effective for symptomatic improvement, but serious complications can occur.

Structure determination of the extracellular xylanase from Geobacillus stearothermophilus by selenomethionyl MAD phasing.

Xylanases are hemicellulases that hydrolyze the internal beta-1,4-glycoside bonds of xylan. The extracellular thermostable endo-1,4-beta-xylanase (EC 3.2.1.8; XT6) produced by the thermophilic bacterium Geobacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids, with a calculated molecular weight of 43 808 Da and a pI of 9.0. Crystallographic studies of XT6 were performed in order to study the mechanism of catalysis and to provide a structural basis for the rational introduction of enhanced thermostability by site-specific mutagenesis. XT6 was crystallized in the primitive trigonal space group P3(2)21, with unit-cell parameters a = b = 112.9, c = 122.7 A. A full diffraction data set for wild-type XT6 has been measured to 2.4 A resolution on flash-frozen crystals using synchrotron radiation. A fully exchanged selenomethionyl XT6 derivative (containing eight Se atoms per XT6 molecule) was also prepared and crystallized in an isomorphous crystal form, providing full selenium MAD data at three wavelengths and enabling phase solution and structure determination. The structure of wild-type XT6 was refined at 2.4 A resolution to a final R factor of 15.6% and an R(free) of 18.6%. The structure demonstrates that XT6 is made up of an eightfold TIM-barrel containing a deep active-site groove, consistent with its 'endo' mode of action. The two essential catalytic carboxylic residues (Glu159 and Glu265) are located at the active site within 5.5 A of each other, as expected for 'retaining' glycoside hydrolases. A unique subdomain was identified in the carboxy-terminal part of the enzyme and was suggested to have a role in xylan binding. The three-dimensional structure of XT6 is of great interest since it provides a favourable starting point for the rational improvement of its already high thermal and pH stabilities, which are required for a number of biotechnological and industrial applications.

Biochemical characterization and identification of catalytic residues in alpha-glucuronidase from Bacillus stearothermophilus T-6.

Alpha-D-glucuronidases cleave the alpha-1,2-glycosidic bond of the 4-O-methyl-D-glucuronic acid side chain of xylan, as a part of an array of xylan hydrolyzing enzymes. The alpha-D-glucuronidase from Bacillus stearothermophilus T-6 was overexpressed in Escherichia coli using the T7 polymerase expression system. The purification procedure included two steps, heat treatment and gel filtration chromatography, and provided over 0.3 g of pure enzyme from 1 L of overnight culture. Based on gel filtration, the native protein is comprised of two identical subunits. Kinetic constants with aldotetraouronic acid as a substrate, at 55 degrees C, were a Km of 0.2 mM, and a specific activity of 42 U x mg(-1) (kcat = 54.9 s(-1)). The enzyme was most active at 65 degrees C, pH 5.5-6.0, in a 10-min assay, and retained 100% of its activity following incubation at 70 degrees C for 20 min. Based on differential scanning calorimetry, the protein denatured at 73.4 degrees C. Truncated forms of the enzyme, lacking either 126 amino acids from its N-terminus or 81 amino acids from its C-terminus, exhibited low residual activity, indicating that the catalytic site is located in the central region of the protein. To identify the potential catalytic residues, site-directed mutagenesis was applied on highly conserved acidic amino acids in the central region. The replacements Glu392-->Cys and Asp364-->Ala resulted in a decrease in activity of about five orders of magnitude, suggesting that these residues are the catalytic pair.

Prevalence of asthma in 13-14 yr-old schoolchildren across Israel.

There is evidence that the incidence and severity of asthma are increasing worldwide, but there are limited data on asthma in Israel. The aim of this study was to investigate the prevalence and severity of asthma and asthma symptoms in 13-14 yr-old schoolchildren in Israel. The self-completed questionnaire used was a modified version of that developed by the International Study of Asthma and Allergies in Childhood (ISAAC), and was administered to a national sample of 12,918 children. The prevalence of asthma ever, wheezing ever and wheezing in the last 12 months were 13.7, 23.8 and 17.9% respectively. Significantly higher rates of a history of asthma and asthma symptoms were observed in Jews compared with Arabs. Although asthma ever was more prevalent in males than in females, asthma symptoms were significantly more common in females. The type of area of residence had no effect on the prevalence of wheezing. The ethnic differences in the prevalence of asthma persisted after controlling for sex, district of residence and level of urbanization. The prevalence of both asthma and asthma symptoms in Israel are slightly above the mean reported from 10 other countries in Europe and the Far-East.

[Use of sentinel reporting clinics for influenza surveillance in the winter of 1996-1997].

In a joint effort of the Israel Center for Disease Control, the National Center for Influenza in the Central Virology Laboratory, together with a group of collaborating pediatricians and family physicians, a network for influenza surveillance was established in the winter of 1996-97. Nose and throat swabs were obtained from 571 patients with flu-like illness. 133 (23%) were positive for influenza virus. Both influenza A(H3N2) and B were isolated, predominantly influenza B during the beginning of the season. Both circulating strains were antigenically similar to those included in the vaccine for 1996-1997. Patients from whom influenza virus was isolated were significantly more likely to suffer from cough and myalgia in comparison with patients whose cultures were negative (p = 0.02 and 0.003, respectively). Results of the first year of surveillance indicate that sentinel reporting clinics are useful for timely detection and identification of the viral strains circulating in the community, thus allowing prompt intervention in preventing the spread of influenza. Conclusions from the first year of the study were drawn and applied in the winter of 1997-1998.

Efficacy of topical gentamicin treatment after 193-nm photorefractive keratectomy in an experimental Pseudomonas keratitis model.

BACKGROUND: The treatment of Pseudomonas keratitis has many limitations, and further investigation to identify more effective approaches is required. We therefore studied the possible contribution of the debridement effect of 193-nm excimer laser on Pseudomonas keratitis in rabbit eyes. METHODS: Pseudomonas keratitis was induced in 30 rabbit eyes by inflicting controlled central corneal scratches and applying a drop of Pseudomonas aeruginosa suspension. After 24 h, one cornea of each animal was photo-ablated (excimer laser: fluency 90 mJ/cm2, 10 Hz, 213 pulses), yielding 50 microns of tissue ablation, while the follow cornea served as control. Five groups of six animals each were formed and received: a subconjunctival injection of gentamicin 20 mg (group 1), topical 14 mg/ml gentamicin hourly (group 2) or every 2.5 h (group 3), or NaCl 0.9% hourly (group 4) for 8 h. In group 5, animals were sacrificed without additional treatment. After 9 h corneas were excised, homogenized, serially diluted, and plated on agar blood plates. The numbers of colony-forming units (CFU) per cornea were statistically evaluated (Mann-Whitney test). RESULTS: In control eyes, a greater decrease of CFU was observed in group 2 than in group 3 (P = 0.03). In laser-ablated eyes, there was no difference in CFU between groups 2 and 3. Comparison of the excimer-treated and control eyes revealed a greater number of bacteria (CFU) in controls only in group 3 (P = 0.02). CONCLUSION: Our study suggests that controlled debridement of cornea with excimer laser may improve the effect of topical antibiotics.

Modal analysis and cut-off conditions of multichannel surface-acoustic-waveguide structures.

Multichannel guides for surface acoustic waves can improve the efficiency of SAW (surface acoustic-wave) devices significantly. Focusing, steering, and modulating the propagating acoustical modes can be achieved similarly to optical waveguided devices. A general formulation is presented for the analysis of the lateral waveguiding properties of Rayleigh modes in surfaces loaded with deposited strips of different materials. General expressions are obtained for the number of modes and cutoff conditions in these structures. As examples of applications, a simple directional coupler and an electrically controlled coupler are proposed.

Patients' reactions to humorous interventions in psychotherapy.

This study explores the preference of patients for humorous or nonhumorous therapist interventions. Hysterical, obsessive, and depressive patients were presented with three functional kinds of humorous interventions: emotional confrontation, anxiety reduction and perspective development. The data indicate that the patients consistently favored the nonhumorous interventions, but the degree of this preference varied according to personality style. Obsessive patients stood out in their ardent repudiation of humorous interventions. The results suggest that the desirability of utilizing humor in therapy depends on a multiplicity of parameters.