Identification of potent and selective inhibitors of PKR via virtual screening and traditional design.

Protein Kinase RNA-activated (PKR) inhibition is thought to be relevant for immunology due to the potential to reduce macrophage and dendritic cell responses to bacteria and its signaling downstream of TNFα. PKR is also associated with neuroscience indications such as Alzheimer's disease due to its activation by the double stranded DNA (dsDNA) virus HSV1, a virus suggested to be important in the development of AD. Studies exploring the mechanistic role of PKR with existing tool molecules such as the tricyclic oxindole C16 are clouded by the poor selectivity profile of this ATP-competitive, Type I kinase inhibitor. Type II kinase leads such as the benzothiophene or pyrazolopyrimidine scaffolds from literature are equally poor in their selectivity profiles. As such, it became necessary to identify more potent and selective chemical matter to better understand PKR biology. A dual approach was taken. The first step of the strategy included virtual screening of the AbbVie compound collection. A combination of pharmacophore-based and GPU shape-based screening was pursued to identify selective chemical matter from promiscuous leads. The second step of the strategy followed traditional compound design. This step initiated from a literature lead with PKR cross reactivity. Combined, the two parallel efforts led to identification of more selective leads for investigation of PKR biology.

The oxidative costs of territory quality and offspring provisioning.

The costs of reproduction are an important constraint that shapes the evolution of life histories, yet our understanding of the proximate mechanism(s) leading to such life-history trade-offs is not well understood. Oxidative stress is a strong candidate measure thought to mediate the costs of reproduction, yet empirical evidence supporting that increased reproductive investment leads to oxidative stress is equivocal. We investigated whether territory quality and offspring provisioning increase oxidative stress in male snow buntings (Plectrophenax nivalis) using a repeated sampling design. We show that arrival oxidative stress is not a constraint on territory quality or the number of offspring fledged. Nevertheless, owners of higher-quality territories experienced an oxidative cost, with this cost increasing more rapidly in younger males. Males that provisioned offspring at a high rate also experienced increased oxidative stress. Together, these findings support the potential role of oxidative stress in mediating life-history trade-offs. Future work should consider that reproductive workload is not limited to offspring care, and other activities - including territory defence - may contribute significantly to the costs of reproduction.

Web-based self-management for patients with multiple sclerosis: a practical, randomized trial.

OBJECTIVE: No studies have addressed the use of electronic personal health records (e-PHRs) for self-management in complex neurological disorders. We assessed and tested an Internet-based self-management system that utilized the e-PHR and determined its impact on self-assessed well-being, clinician-assessed well-being, and healthcare utilization in patients with multiple sclerosis (MS). MATERIALS AND METHODS: Subjects were randomized to usual care (a secure Web-based messaging system) or active intervention, which included secure messaging, self-monitoring, self-management of MS symptoms, and communication about upcoming clinic visits. Computers and Internet access were provided. Subjects were included if they had MS, lived within the county or region surrounding our MS center, had at least two appointments at our center in the previous 12 months, and demonstrated basic typing and computer skills. Study duration was 12 months. RESULTS: Of 220 subjects completing informed consent, 206 met the inclusion criteria. At the study's end, 83 subjects remained in the usual care group and 84 in the enhanced care group. Both groups used the available system components. The groups did not significantly differ on the primary endpoints or healthcare utilization. CONCLUSIONS: Self-management support is an emerging aspect of chronic care management. We established the feasibility of conducting a randomized, controlled trial using e-PHRs for patient self-management. We did not find that e-PHR-enabled self-management augmented multidisciplinary MS center-based care, possibly because the differences between interventions were not great enough.

Supranuclear eye movements and nystagmus in children: A review of the literature and guide to clinical examination, interpretation of findings and age-appropriate norms.

Abnormal eye movements in children, including nystagmus, present a significant challenge to ophthalmologists and other healthcare professionals. Similarly, examination of supranuclear eye movements and nystagmus in children and interpretation of any resulting clinical signs can seem very complex. A structured assessment is often lacking although in many cases, simple clinical observations, combined with a basic understanding of the underlying neurology, can hold the key to clinical diagnosis. As the range of underlying diagnoses for children with abnormal eye movements is broad, recognising clinical patterns and understanding their neurological basis is also imperative for ongoing management. Here, we present a review and best practice guide for a structured, methodical clinical examination of supranuclear eye movements and nystagmus in children, a guide to clinical interpretation and age-appropriate norms. We also detail the more common specific clinical findings and how they should be interpreted and used to guide further management. In summary, this review will encourage clinicians to combine a structured assessment and a logical interpretation of the resulting clinical signs, in order to recognise patterns of presentation and avoid unnecessary investigations and protracted delays in diagnosis and clinical care.

The spectral main sequence of human saccades.

Despite the many models of saccadic eye movements, little attention has been paid to the shape of saccade trajectories. Some investigators have argued that saccades are driven by a rectangular "bang-bang" neural control signal, whereas others have emphasized the similarity to fast arm movement trajectories, such as the "minimum jerk" profile. However, models have not been tested rigorously against empirical trajectories. We examined the Fourier transforms of saccades and compared them with theoretical models. Horizontal saccades were recorded from 10 healthy subjects. The Fourier transform of each saccade was accurately computed using a padded fast Fourier transform (FFT), and the frequencies of the first three minima (M1, M2, M3) in each energy spectrum were measured to a precision of 0.12 Hz. Each subject showed near-linear trends in the relationships among M1, M2, and M3 and the reciprocal of duration (1/T), which we call the "spectral main sequence." Extrapolation of plots did not pass through the origin, indicating a subtle departure from self-similarity. Bivariate confidence regions were established to allow for slope-intercept variability. The nonharmonic relationships seen cannot arise from a rectangular saccadic pulse driving a linear ocular plant. The relationships are also incompatible with minimum acceleration, minimum jerk, or higher-order minimum square derivative trajectories. The best fits were made by trajectories that minimize postmovement variance with signal-dependent noise (). It is concluded that the spectral main sequence is exquisitely sensitive to the saccade trajectory and should be used to test objectively all present and future models of saccades.

Band-shape analysis and resolution of electronic spectra of pyridoxal phosphate and other 3-hydroxypyridine-4-aldehydes.

The electronic absorption spectra of individual ionic forms of pyridoxal phosphate and of a series of related aldehydes have been evaluated together with pKa values. Spectral resolution with lognormal curves has permitted the quantitative description of equilibria for hydration and tautomerization. Precise values of peak positions for both aldehyde and hydrate forms have been obtained. Measurements of temperature-induced changes in the spectra have provided additional information. Knowing the hydration ratios and stepwide acid dissociation constants, it is possible to evaluate microscopic acid dissociation constants for both the aldehyde and hydrate forms of the compounds.

Familial vestibulocerebellar disorder maps to chromosome 13q31-q33: a new nystagmus locus.

PURPOSE: To determine a gene locus for a family with a dominantly inherited vestibulocerebellar disorder characterised by early onset, but not congenital nystagmus. DESIGN: Observational and experimental study. METHODS: We carried out a phenotypic study of a unique four generation family with nystagmus. We performed genetic linkage studies including a genome wide search. RESULTS: Affected family members developed vestibulocerebellar type nystagmus in the first two years of life. A higher incidence of strabismus was noted in affected members. Haplotype construction and analysis of recombination events linked the disorder to a locus (NYS4) on chromosome 13q31-q33 with a lod score of 6.322 at theta=0 for D13S159 and narrowed the region to a 13.8 cM region between markers D13S1300 and D13S158. CONCLUSIONS: This study suggests that the early onset acquired nystagmus seen in this family is caused by a single gene defect. Identification of the gene may hold the key to understanding pathways for early eye stabilisation and strabismus.

Effect of lecithin on memory in normal adults.

In a double-blind, placebo-controlled, crossover study of the effect of lecithin on memory test performance, nine normal paid volunteers (age range = 22-55 years) took single oral doses of placebo and lecithin (20 g) 5 hours before cognitive testing. Lecithin raised plasma choline levels during the test session to nearly double the placebo levels, but a categorized serial learning task, a word recognition task, a paired associates learning task, and a test of retrieval by category demonstrated no significant change in memory performance. Lecithin did not selectively enhance memory for low-imagery words. Lecithin also had no effect on psychomotor speed.

Identification and role of ionizing functional groups at the active center of Rhodotorula gracilis D-amino acid oxidase.

D-Amino acid oxidase (DAAO) is a flavoprotein oxidase that catalyzes the oxidation of amino acids and produces ketoacids and H(2)O(2). The rate of product release from reduced DAAO from Rhodotorula gracilis is pH dependent and reflects a pK(a) of approximately 9.3. Binding of benzoate and 3,3,3-trifluoro-D-alanine to wild-type and Y238F-DAAO is also pH dependent (pK(a)=9.8+/-0.1 and 9.05+/-0.1, respectively for benzoate binding). However, binding of benzoate to Y223F-DAAO is pH independent, indicating the pK(a) is due to Y223-OH. This latter residue is thus involved in substrate binding, and probably is the group that governs product release. In contrast to this, the second active site tyrosine, Y238, has little influence on ligand binding.

Differences in ambulatory test ordering in England and America. Role of doctors' beliefs and attitudes.

Forty-five American and English doctors were surveyed to determine whether differences in their beliefs about the incidence and morbidity of complications and the role of testing for patients with chronic hypertension could be contributing to the large cross-national differences previously demonstrated in ambulatory test use for such patients. For each of nine tests, the number the English doctors thought they "should order" was significantly less than that of the American group. English doctors also estimated a lower incidence for each of seven possible complications of hypertension, but there was no difference in perceived morbidity of the complications. Both groups thought results from testing would alter therapy for only a small proportion of patients; however, the English estimate was significantly smaller than the American (16 percent versus 27 percent: p less than 0.05). The reasons given for testing were very similar in the two countries except that American doctors ranked as more important the reason that patients used the quantity of test ordering as an indicator of quality care. These results suggest that differences in ambulatory test use are consistent with physicians' beliefs about the number of tests they should be ordering, disease incidence, the likelihood that testing will affect patient care, and patient expectations. Further study should be directed toward understanding the contributions that differences in physician beliefs about the natural histories of diseases and patient expectations make to variations in test ordering.

Antithrombin III prophylaxis of venous thromboembolic disease after total hip or total knee replacement.

The use of antithrombin III (ATIII) replacement in combination with low-dose heparin therapy in prevention of postoperative venous thrombosis in patients following total hip replacement or total knee replacement was evaluated. A randomized prospective venographically controlled trial in hip replacement compared treatment with dextran 40 with a regimen of ATIII (1,500 units preoperatively and 1,000 units daily for five days) and low-dose heparin. Patients receiving ATIII/heparin had significantly higher postoperative ATIII concentrations than dextran-treated patients and also had a low incidence of venous thromboembolic disease (7 percent). The ATIII/heparin regimen was well tolerated with no increase in bleeding or significant prolongation of the activated partial thromboplastin time. Two cohorts of patients undergoing total knee replacement were studied using different doses of ATIII in combination with heparin. An initial 10 patients were treated with the same ATIII dose used for patients undergoing total hip replacement, with a 50 percent incidence of venous thrombosis. A second group of 11 patients was treated with twice the dose of ATIII, and an incidence of venous thrombosis of 27 percent was found. The higher ATIII dose resulted in significantly higher ATIII concentrations and maintained the postoperative ATIII concentration above normal. Among patients receiving prophylaxis with either warfarin. dextran, or ATIII/heparin, no clear association was found between reduced ATIII concentrations and occurrence of venous thrombosis. It is concluded that ATIII replacement following total hip or knee replacement corrects the postoperative ATIII deficiency and that the combination of ATIII and low-dose heparin is an effective prophylactic regimen following total hip replacement.

Anxiety-like subjective effect of ethanol antagonist RO 15-4513 demonstrated in pentylenetetrazol discrimination.

Ro 15-4513, a benzodiazepine-receptor ligand which antagonizes ethanol, was tested in the pentylenetetrazol discrimination, a bioassay for anxiogenic drugs. Rats were trained with food reward to discriminate pentylenetetrazol (PTZ) from saline in a two-lever operant task. In lever-selection tests, rats selected the PTZ lever both after PTZ and after Ro 15-4513. The PTZ-like stimulus produced by Ro 15-4513 was blocked by diazepam and by the benzodiazepine receptor blocker Ro 15-1788. Substitution for the anxiogenic drug PTZ, and blockade by the anxiolytic diazepam, support the hypothesis that Ro 15-4513 is anxiogenic; blockade by Ro 15-1788 suggests that the PTZ-like stimulus produced by Ro 15-4513 occurs through its action at the benzodiazepine receptor.

Comparison of two warfarin regimens in the prevention of venous thrombosis following total knee replacement.

A prospective, randomized trial was conducted to compare the effectiveness and safety of warfarin given in two regimens in prevention of venous thrombosis after total knee replacement. Adult patients scheduled for primary or revision total knee replacement were randomly assigned to receive either a "two-step" warfarin regimen beginning 10-14 days pre-operatively or, alternatively, to begin warfarin the night before surgery. Post-operatively, the dose was adjusted in both groups to achieve a target International Normalized Ratio (INR) of 2.2 and prophylaxis was continued until venography on post-operative days five through nine. Bleeding was assessed by surgical blood loss, transfusion requirements, changes in hematocrit, and clinically identified bleeding complications. The occurrence of deep vein thrombosis was nearly the same in the two treatment groups, 39% in patients randomized to the two-step regimen as compared to 38% in those beginning the night before surgery. The occurrence of proximal vein thrombosis was also similar, 5% versus 7% (p = NS). Patients in the two-step group received 1.33 +/- 1.26 transfusions compared to 0.95 +/- 1.22 in the night before group (p < 0.05) and also had a lower nadir post-operative hematocrit of 26.7 +/- 3.1 as compared to 28.5 +/- 3.2 (p < 0.0001). Major bleeding complications were associated with excessively prolonged INRs and occurred in five patients in the two-step group and two in the night before group. Patients in both groups who developed thrombosis had a significantly lower INR on post-operative days two and three compared to those without thrombosis. We conclude that a prophylactic warfarin regimen for prevention of deep vein thrombosis after total knee replacement beginning the night before surgery is more convenient and may be associated with less bleeding than a regimen beginning warfarin 10-14 days pre-operatively. Careful control of anticoagulant intensity is needed to achieve maximum effectiveness and avoidance of bleeding complications.

The prevalence and prophylaxis of gout in England.

The prevalence of gout in England was reported as having nearly doubled in the 1970s to about 3 per 1000, and it is possible that it has continued to increase. There may also have been some change in the level of prophylactic therapy compared with the 44.8% recorded in 1979. In this study, data were provided from their morbidity registers by 40 volunteer practices with a combined population of over 300,000 patients. Analyses of prevalence by age and sex, and of the extent of prophylaxis were made. The male:female ratio and the increase with age found were in line with earlier work, but overall prevalence was much higher at almost 10 per 1000. There was considerable inter-practice variation: 20% of this could be accounted for by the age and sex structure of the practice populations, but neither economic status nor broad geographical factors appeared to be significant, and it did not appear to be due to variation between practices in the level of special interest in gout. Prophylactic therapy--almost entirely allopurinol--was being prescribed for 48% of the sufferers; though practices varied widely in their propensity to give it, as a group the amount of allopurinol they used was close to the national average. The increase found in the prevalence of gout may be related to an increase in obesity in the population.

Sensitivity of pentylenetetrazol discrimination increased by a stimulus fading technique.

The interoceptive stimulus produced by pentylenetetrazol (PTZ) is pharmacologically similar to anxiety and is used in a behavioral assay for anxiety-related stimuli (the PTZ model of anxiety). The stimulus fading technique was tested as a method to increase the sensitivity of this assay. Rats were trained with food-reward to press one lever after injection of PTZ and an alternate lever after saline. Rats initially learned the discrimination at a PTZ dose of 20 mg/kg. They were then trained with sequentially lower doses until they reliably discriminated a PTZ dose of 10 mg/kg. Substitution tests with other doses and drugs showed that, after the fading procedure, dose-response curves were shifted to lower doses for PTZ, Ro 5-3663, and nicotine Similarly, the dose of diazepam required to block the low dose of PTZ was lower than that required to block the higher dose of PTZ. These results indicated that the sensitivity of the discrimination was enhanced in rats trained to discriminate a lower dose of PTZ. Doses of nikethamide, cocaine, and yohimbine that did not substitute for the higher dose of PTZ also did not substitute for the lower dose. These data suggest that rats can be trained to discriminate a low dose of PTZ by the stimulus fading technique. Moreover, they suggest that this training method does not compromise the specificity of the discrimination.

Quantal detection and homogeneous sensitivity in a pentylenetetrazol discrimination.

Rats were trained to discriminate pentylenetetrazol (PTZ, 20 mg/kg) from saline in a two-lever operant task. Correct lever presses were reinforced with food under the control of a fixed ratio 10 schedule. In tests of the effect of PTZ dose on lever selection, rats selected the PTZ lever in a dose-dependent manner, with peak latency at the approximate ED50 dose (10 mg/kg). Rats usually pressed only the selected lever, regardless of dose, indicating that lever selection was a quantal (or bimodal) function of stimulus intensity. Lever biases observed during training sessions did not predict the performance of individual rats in tests with the ED50 dose. In three independent trials with this intermediate dosage, the rats selecting the PTZ lever varied from trial to trial, suggesting that rats detecting this dose did not form a stable subgroup. The pattern of lever selections across these three trials was not significantly different from that predicted by a model in which all subjects shared the same probability for detecting the drug stimulus. These results demonstrate that lever selection in a two-lever drug-discrimination task can be quantal in nature, and suggest that rats trained with PTZ, 20 mg/kg, are homogeneous in sensitivity to this stimulus.

Withdrawal from chronic nicotine substitutes partially for the interoceptive stimulus produced by pentylenetetrazol (PTZ).

Rats were trained on an FR10 schedule of food reinforcement to press one lever after pentylenetetrazol (PTZ), 20 mg/kg, IP, and an alternate lever after saline. After acute nicotine, 0.64 mg/kg, SC, 35% of the rats pressed the PTZ-lever. Diazepam, 5 mg/kg, IP, blocked the stimulus produced by PTZ, and mecamylamine, 5 mg/kg, IP, blocked the stimulus produced by nicotine. Training was then suspended and rats were treated with nicotine, at 8-h intervals, 0.64 mg/kg on the 1st day, and 1.25 mg/kg on subsequent days, for 21 days. To determine whether nicotine withdrawal substitutes for the stimulus produced by PTZ, rats were tested with saline at various times after chronic nicotine injections. Data from this part of the study were replicated in another group given nicotine for 15 days. Saline at 8 h after nicotine (five determinations each group) produced a small but stable degree of PTZ lever selection (35 +/- 4%). At 48 h after termination of nicotine treatment, the percentage of rats selecting the PTZ lever (50%) was greater than that in a control group tested after an equivalent period without training. The PTZ-like stimulus detected after chronic nicotine was not altered by mecamylamine, was additive with PTZ, and was blocked by diazepam. These data suggest that withdrawal from chronic nicotine produces a weak PTZ-like stimulus, which can be antagonized by an anxiolytic drug.

A method to shorten the training phase of drug discrimination.

Rats were trained to discriminate "drug" from "no drug" in a two-lever, food-reinforced task. One group was trained with cocaine (10 mg/kg) and a second group was trained with pentylenetetrazol (20 mg/kg). A method designed to shorten the time required for the training phase of drug discrimination experiments was assessed in subgroups for each drug. In one subgroup, single training sessions were conducted daily. In the other subgroup, a second session (either drug or saline) was conducted on days for which the first condition was saline. The training conditions were presented in an irregular sequence, with the same condition occurring in no more than two consecutive sessions. Rats trained by the accelerated method learned the discrimination in fewer days, with no decrement in acquisition per session, suggesting that drug discrimination training can be accomplished more rapidly by reducing inter-session interval.

Withdrawal from morphine generalizes to a pentylenetetrazol stimulus.

Rats trained to discriminate pentylenetetrazol (PTZ) from saline in a two-lever food-reinforced operant task were given a three-day course of morphine, 15 to 45 mg/kg tid, ip. On the third day naloxone produced dose-dependent generalization to the PTZ stimulus, with 66% of subjects selecting the PTZ lever after the highest dose (0.32 mg/kg). Following termination of morphine injections, generalization of spontaneous withdrawal was tested. Approximately 50% of subjects selected the PTZ lever at 24 and 48 hrs after the last morphine, and by 96 hrs the percentage of subjects selecting the PTZ lever had dropped to 11%. Rats that chose the PTZ lever at 48 hrs were given diazepam, 5.0 mg/kg, which blocked the PTZ-like stimulus. These data demonstrate that morphine withdrawal produces a stimulus with PTZ-like characteristics which can be blocked by an anxiolytic, and they suggest that the PTZ discrimination may have general utility for investigating drug dependence and withdrawal in animals.

The Fourier analysis of biological transients.

With modern computing technology the digital implementation of the Fourier transform is widely available, mostly in the form of the fast Fourier transform (FFT). Although the FFT has become almost synonymous with the Fourier transform, it is a fast numerical technique for computing the discrete Fourier transform (DFT) of a finite sequence of sampled data. The DFT is not directly equivalent to the continuous Fourier transform of the underlying biological signal, which becomes important when analyzing biological transients. Although this distinction is well known by some, for many it leads to confusion in how to interpret the FFT of biological data, and in how to precondition data so as to yield a more accurate Fourier transform using the FFT. We review here the fundamentals of Fourier analysis with emphasis on the analysis of transient signals. As an example of a transient, we consider the human saccade to illustrate the pitfalls and advantages of various Fourier analyses.