High-throughput screen for sorting cells capable of producing the biofuel feedstock botryococcene.

Botryococcene is a branched triterpene produced by the algae Botryococcus braunii. Hydrocracking botryococcene yields a variety of combustible fuels such as gasoline and jet fuel. Engineering host systems and proteins involved in the biosynthesis of botryococcene to optimize production is of interest given these applications. The current study investigates the use of a diaryltetrazole based screen that undergoes a photoclick reaction with terminal alkenes, such as the branched terminal alkene present on botryococcene, to yield a fluorescent product. Host E. coli systems were established to produce botryococcene, squalene, and no triterpene to serve as a control. Cells were incubated with tetrazole and briefly irradiated with UV light to initiate the photoclick reaction. It was found that the botryococcene producing cells yielded observable fluorescence while the squalene and control cells had negligible fluorescence turn-on activity. Fluorescence-activated cell sorting (FACS) was subsequently used to identify and sort botryococcene producing E. coli from a mixture of control and squalene producing cells.

Studies with Guanidinium- and Amidinium-Based Inhibitors Suggest Minimal Stabilization of Allylic Carbocation Intermediates by Dehydrosqualene and Squalene Synthases.

Dehydrosqualene and squalene synthases catalyze the redox neutral and the reductive, head-to-head dimerization of farnesyl diphosphate, respectively. In each case, the reaction is thought to proceed via an initial dissociation of farnesyl diphosphate to form an allylic carbocation-pyrophosphate ion pair. This work describes the synthesis and testing of inhibitors in which a guanidinium or amidinium moiety is flanked by a phosphonylphosphinate group and a hydrocarbon tail. These functional groups bear a planar, delocalized, positive charge and therefore should act as excellent mimics of an allylic carbocation. An inhibitor bearing a neutral urea moiety was also prepared as a control. The positively charged inhibitors acted as competitive inhibitors against Staphylococcus aureus dehydrosqualene synthase with Ki values in the low micromolar range. Surprisingly, the neutral urea inhibitor was the most potent of the three. Similar trends were seen with the first half reaction of human squalene synthase. One interpretation of these results is that the active sites of these enzymes do not directly stabilize the allylic carbocation via electrostatic or π-cation interactions. Instead, it is likely that the enzymes use tight binding to the pyrophosphate and lipid moieties to promote catalysis and that electrostatic stabilization of the carbocation is provided by the bound pyrophosphate product. An alternate possibility is that these inhibitors cannot bind to the "ionization FPP-binding site" of the enzyme and only bind to the "nonionizing FPP-binding site". In either case, all reported attempts to generate potent inhibitors with cationic FPP analogues have been unsuccessful to date.

A generalized estimating equation approach to analysis of maintenance of wakefulness testing in a study of lisdexamfetamine dimesylate, armodafinil, and placebo in sleep-deprived adults.

In a study of acute sleep deprivation in healthy male volunteers randomized to double-blind treatment with lisdexamfetamine dimesylate (20, 50, or 70 mg), placebo control, or an active control (armodafinil 250 mg), Maintenance of Wakefulness Test data were compared using a generalized estimating equation analysis to eliminate the need for unequivocal sleep latency imputation. Compared with placebo across all Maintenance of Wakefulness Tests, all active treatments were associated with lower risk of falling asleep (risk ratio [95% confidence interval]): 0.45 (0.27-0.76; P = 0.0026), 0.10 (0.05-0.20; P < 0.0001), and 0.05 (0.02-0.14; P < 0.0001) for 20, 50, and 70 mg lisdexamfetamine dimesylate, respectively, and 0.11 (0.06-0.21; P < 0.0001) for the active control. Sleep-risk ratios were similar for lisdexamfetamine dimesylate 50 or 70 mg and for the active control, but lisdexamfetamine 20 mg was associated with a greater risk of falling asleep compared with the active control (4.13 [1.97-8.67]; P = 0.0002). Generalized estimating equation analysis detected wake-promoting effects of active treatments and eliminating data imputation, suggesting model utility in future studies.

Maintenance of wakefulness with lisdexamfetamine dimesylate, compared with placebo and armodafinil in healthy adult males undergoing acute sleep loss.

This study evaluated daytime alertness and performance with lisdexamfetamine dimesylate during acute sleep loss. In a randomized, double-blind study in healthy adult men (n = 135) undergoing 24-hour sleep loss, the alerting effects of single oral lisdexamfetamine dimesylate doses (20, 50, or 70 mg) were compared with a placebo and an active control (armodafinil 250 mg). Primary end point was mean unequivocal sleep latency on the 30-minute maintenance of wakefulness test taken every 2 hours from midnight to 8:00 A.M. Secondary end points included the Karolinska sleepiness scale and psychomotor vigilance task. Safety assessments included treatment-emergent adverse events (TEAEs) and vital signs. Least squares mean (SE) maintenance of wakefulness test unequivocal sleep latency (in minutes) was longer with lisdexamfetamine dimesylate 20, 50, and 70 mg, or armodafinil 250 mg (23.3 [1.10], 27.9 [0.64], 29.3 [0.44], or 27.6 [0.63], respectively) versus placebo (15.3 [1.00]; P < 0.0001). Longer mean unequivocal sleep latency was seen with lisdexamfetamine dimesylate 70 mg versus armodafinil (P = 0.0351) and armodafinil versus lisdexamfetamine dimesylate 20 mg (P = 0.0014). On Karolinska sleepiness scale, lisdexamfetamine dimesylate 50 and 70 mg improved estimated sleepiness versus placebo (P ≤ 0.0002) and armodafinil (P ≤ 0.03). Active treatments improved psychomotor vigilance task performance versus placebo (P < 0.0001). The TEAEs were mild/moderate. No serious adverse events occurred. The most common TEAE was headache with lisdexamfetamine dimesylate and armodafinil (7.4% each) versus placebo (3.7%). Small mean increases in vital signs were observed with lisdexamfetamine dimesylate and armodafinil. In sleep-deprived healthy men, alertness was greater with lisdexamfetamine dimesylate and armodafinil versus placebo on the primary end point. Studies are needed in clinical populations and using longer durations of administration.

Guidelines for the recording and evaluation of pharmaco-sleep studies in man: the International Pharmaco-EEG Society (IPEG).

The International Pharmaco-EEG Society (IPEG) presents guidelines summarising the requirements for the recording and computerised evaluation of pharmaco-sleep data in man. Over the past years, technical and data-processing methods have advanced steadily, thus enhancing data quality and expanding the palette of sleep assessment tools that can be used to investigate the activity of drugs on the central nervous system (CNS), determine the time course of effects and pharmacodynamic properties of novel therapeutics, hence enabling the study of the pharmacokinetic/pharmacodynamic relationship, and evaluate the CNS penetration or toxicity of compounds. However, despite the presence of robust guidelines on the scoring of polysomnography -recordings, a review of the literature reveals inconsistent -aspects in the operating procedures from one study to another. While this fact does not invalidate results, the lack of standardisation constitutes a regrettable shortcoming, especially in the context of drug development programmes. The present guidelines are intended to assist investigators, who are using pharmaco-sleep measures in clinical research, in an effort to provide clear and concise recommendations and thereby to standardise methodology and facilitate comparability of data across laboratories.

Monomer-dimer control and crystal engineering in TASPs.

We have reported a template assembled synthetic protein (cavitein Q4) as an unexpected dimer in the solid state and as a monomer-dimer equilibrium in solution. We have since reported an ability to bias a cavitein's monomer-dimer equilibrium in solution by sequence design involving histidine metal chelation or disulfide incorporation. However, little remains known about the forces contributing to dimeric cavitein crystal nucleation and lattice stabilization. We, therefore, designed glutamine variants to probe factors involved in dimeric cavitein crystallization. It was found that a key glutamate hydrogen-bonding interaction between dimers is integral to crystal formation and stabilization. Additionally, we obtained a crystal structure of a cavitein (Q4-E3H) designed to bias the dimeric structure via histidine metal coordination. The resolved structure indicates a histidine cluster interaction that likely accounts for the biased dimeric form observed in solution.

Conformationally constrained sequence designs to bias monomer-dimer equilibriums in TASP systems.

We have designed template-assembled synthetic proteins (TASPs) with the intent of controlling their oligomeric state by stabilizing specific helical tertiary structures via histidine metal ion chelation or disulfide incorporation. In solution, cavitein Q4 was previously determined to interconvert between a four-helix bundle monomer and an eight-helix bundle dimer. In this paper, we show that judicious mutation of cavitein Q4 can stabilize either the monomeric parallel four-helix bundle or the dimeric antiparallel eight-helix bundle structure. Cavitein Q4-E3H, designed to be dimeric, is indeed biased toward dimerization as a result of incorporation of histidines. Moreover, the addition of nickel was found to further increase the association constant of dimerization. Similarly, a cavitein designed to stabilize the monomeric structure via histidine metal ion chelation (Q4-H) was found to favor a monomer in solution upon addition of nickel. Lastly, a cavitein intended to stabilize a monomeric structure via disulfide incorporation (Q4-C2) is reported. Surprisingly, this disulfide cavitein yielded two products upon oxidation suggesting disulfide formation both above the cavitand template and below may be possible. Nevertheless, the two disulfide caviteins were shown to exist as monomers as per their design.

A randomized phase 1 single-dose polysomnography study of ASP8062, a GABAB receptor positive allosteric modulator.

RATIONALE: Previous research suggests that sleep polysomnography and EEG endpoints can be used to assess GABAergic activity; however, the impact of GABAB receptor positive allosteric modulators on sleep endpoints remains unclear. OBJECTIVES: This phase 1 study compared a single dose of ASP8062 (35 mg or 70 mg), a GABAB receptor positive allosteric modulator, with placebo and paroxetine (40 mg). METHODS: Healthy adult volunteers were randomized to four treatments (35 mg ASP8062, 70 mg ASP8062, paroxetine 40 mg, or matching placebo), each separated by a 14-day washout. Primary endpoints obtained by polysomnography were time in stage N3 or SWS and time in rapid eye movement (REM) sleep. Secondary endpoints included impact on sleep stages and electroencephalography parameters, pharmacokinetics, nighttime growth hormone (GH), and safety/tolerability. RESULTS: In 20 randomized volunteers, ASP8062 led to a significant and seemingly dose-dependent increase in SWS over the entire night; this increase was mainly observed during the first third of the night. ASP8062 did not impact time in REM sleep. Paroxetine had no effect on SWS but produced a significant reduction in time spent in REM sleep. A dose-dependent trend in increased GH release was also observed with ASP8062. Headache and nausea were the most commonly reported treatment-emergent adverse events (TEAEs) for ASP8062; most TEAEs were mild in severity. CONCLUSIONS: Single-dose ASP8062 (35 and 70 mg) appeared to result in CNS penetration and enhanced GABAergic activity as measured by increases in slow-wave sleep and growth hormone release.

Maskless photolithography via holographic optical projection.

We propose an inexpensive novel rapid prototyping approach to a maskless and fully adaptive photolithographic process. Phase-only computer-generated holograms of lithographic masks displayed on a liquid-crystal-on-silicon spatial light modulator were used in a holographic optical lithography system. Using holographic projection allows diffraction-limited performance within the given parameters of the optical system, adaptive software refocusing, and a continuous, pixel-free pattern. With the demonstrator, we have successfully proven the concept for micrometer-size lithographic features.

X-ray crystal analysis of a TASP: structural insights of a cavitein dimer.

Cavitein Q4 is a template assembled synthetic protein designed for X-ray crystallographic analysis. It is based on a previous monomeric helical bundle cavitein (N1GG) that consists of four identical parallel helical peptides. Crystals that were grown in the presence of bromide ions were used to solve the initial phases via single-wavelength anomalous dispersion (SAD). A 1.4 A resolution data set was then refined starting with the SAD phases to provide the crystal structure of cavitein Q4. The crystal structure revealed cavitein Q4 as an asymmetric dimer, although the cavitein appears to be largely monomeric in solution. A comparative analysis is carried out to discern any intrinsic differences between Q4 and its parent cavitein N1GG. We present herein the first X-ray crystal structure of a TASP system and relate this structure to the solution data for both Q4 and its parent N1GG.

High speed liquid crystal over silicon display based on the flexoelectro-optic effect.

One of the key technologies to evolve in the displays market in recent years is liquid crystal over silicon (LCOS) microdisplays. Traditional LCOS devices and applications such as rear projection televisions, have been based on intensity modulation electro-optical effects, however, recent developments have shown that multi-level phase modulation from these devices is extremely sought after for applications such as holographic projectors, optical correlators and adaptive optics. Here, we propose alternative device geometry based on the flexoelectric-optic effect in a chiral nematic liquid crystal. This device is capable of delivering a multilevel phase shift at response times less than 100 microsec which has been verified by phase shift interferometry using an LCOS test device. The flexoelectric on silicon device, due to its remarkable characteristics, enables the next generation of holographic devices to be realized.

Future of Health: Findings from a survey of stakeholders on the future of health and healthcare in England.

This article presents findings from a survey conducted by RAND Europe at the request of the National Institute for Health Research (NIHR) to gather and synthesise stakeholder views on the future of health and healthcare in England in 20 to 30 years' time. The aim of the research was to generate an evidenced-based picture of the future health and healthcare needs, and how it might differ from today, in order to inform strategic discussions about the future priorities of the NIHR and the health and social care research communities more broadly. The survey provided a rich and varied dataset based on responses from 300 stakeholders in total. A wide range of fields were represented, including public health, social care, primary care, cancer, genomics, mental health, geriatrics, child health, patient advocacy and health policy. The respondent group also included a number of professional and private stakeholder categories, such as clinicians, policy experts, academics and patient and public representatives. The study findings validate a number of prominent health research priorities currently visible in England, such as antimicrobial resistance, the burden of dementia and age-related multi-morbidity, digital health and genomics. Interest in these areas and other themes, such as mental health, health inequalities and transforming health service models, cut across multiple disciplinary boundaries. However, it is clear that there are a variety of views among stakeholders on the relative importance of these areas of focus, and the best approach to manage their emergence in the coming decades. The full dataset of survey responses, for which permission to share was given, is a useful resource for those seeking to engage with a particular issue in more depth. The dataset can be found on NIHR's website at: http://nihr.ac.uk/news-and-events/documents/quotes.xls.

Dynamics and kinetics of a modified-release formulation of zolpidem: comparison with immediate-release standard zolpidem and placebo.

Modified-release (MR) zolpidem was developed to maintain effective plasma concentrations during the 3- to 6-hour post-dosage interval, corresponding to the middle portion of the typical sleep interval. Modified-release zolpidem (12.5 mg), standard immediate-release (IR) zolpidem (10 mg), and placebo were compared in a double-blind, single-dose, 3-way crossover daytime study of healthy volunteers (n = 70 completers). Effect areas for electroencephalographic beta amplitude during 0 to 8 hours and 3 to 6 hours after dosage were greater for MR compared to IR (P < .001). The digit-symbol substitution test and sedation rating scales behaved similarly. MR and IR did not differ in effects at 8 hours post-dosage nor in halflife or clearance. Time of peak plasma concentration (tmax) was significantly longer for MR (2.4 vs 2.0 hours, P < .004), and dose-normalized peak plasma concentration (Cmax) was lower (12.2 vs 14.0 ng/mL/mg, P < .001). MR zolpidem also had greater area under the plasma concentration curve (AUC) during the 3- to 6-hour interval (P < .001). Thus, MR zolpidem produces sustained plasma levels compared to IR, with resulting enhancement of pharmacodynamic effects in the 3- to 6-hour post-dosage interval.

Comparison of actigraphy and polysomnography to assess effects of zolpidem in a clinical research unit.

OBJECTIVE: This study sought to compare devices that use actigraphy for measuring sleep endpoints in the clinical research unit (CRU) and home environment. The abilities of polysomnography (PSG) and actigraphy monitors to detect drug effects in a CRU were also investigated. METHODS: Eleven healthy subjects were recruited and monitored with PSG for four consecutive nights in a CRU after receiving no treatment (night 1, N1), and then placebo or 5 mg day(-1) or 10 mg day(-1) zolpidem in a randomised, cross-over design. Subjects wore two devices that use actigraphy (a Respironics® Actiwatch® on the wrist and a BodyMedia® Sensewear® Armband on the upper-arm) on the non-dominant arm for five nights at home and four nights in the CRU during PSG. RESULTS: Wake after sleep onset (WASO) and total sleep time (TST) measured by PSG and estimates of WASO by the Actiwatch decreased significantly with 5mg but not 10mg of zolpidem versus placebo. Direct activity (counts/min) with the Actiwatch decreased in response to zolpidem (both 5 and 10 mg day(-1)) versus placebo. Armband recordings of direct activity were similar to the Actiwatch but not significantly different versus placebo. Both actigraphy device estimates of TST were approximately 1h longer in CRU versus home. Agreement between actigraphy estimates of TST and WASO and PSG values of TST and WASO were closer during nights with zolpidem treatment. CONCLUSIONS: PSG can detect the effects of zolpidem on sleep in a CRU setting. Actigraphy can provide useful assessment of sleep, but direct activity endpoints may be more effective than estimates of TST and WASO.

Four-helix bundle cavitein reveals middle leucine as linchpin.

A template-assembled de novo four-helix bundle is used to examine the hydrophobic effect within the bundle interior. Leu to Ala variants of the basis sequence GG-EELLKKLEELLKKG were characterized by GuHCl denaturation, NMR dispersion, and N-H/D exchange experiments. The results show that the middle leucine (L7) is imperative in maintaining bundle stability. The average leucine was found to contribute 1.8 kcal mol(-1) toward stability, whereas the middle leucines contribute 2.7 kcal mol(-1) each. Substituting alanine into the middle position (7) constitutes a striking 95% reduction of the overall cavitein stability.

The efficacy of ECT in adults with mental retardation experiencing psychiatric disorders.

There is a paucity of empirical data establishing the efficacy of electroconvulsive therapy (ECT) in patients with mental retardation and psychiatric disorders. This study examines the efficacy of ECT on specific symptoms and between psychiatric diagnoses in patients with mental retardation who are psychiatrically ill. A chart review was performed on 20 inpatients who had received ECT on a dedicated Mental Retardation-Dual Diagnosis Unit and were divided into 3 categories: mood disorders (n = 12), psychotic disorders (n = 6), and intermittent explosive disorder (n = 2). Ratings were performed 1 week before ECT treatment and 1-week after its termination using the Aberrant Behavior Checklist and the Clinical Global Impressions Severity Scale. A repeated-measures analysis of variance comparing Aberrant Behavior Checklist scale scores revealed a significant time-by-treatment interaction (F = 75.43, df = 1,9, P = 0.000, 2 t). The mood disorder and psychotic disorder groups had significantly lower irritability and hyperactivity scores after treatment compared with the intermittent explosive disorder group. The Clinical Global Impressions Severity Scale rating scores showed significant improvement in the mood disorders group (67%), in contrast to the intermittent explosive disorder group (0%). Our data suggests the utility of ECT for patients with mental retardation who also have treatment-resistant mood disorders and psychotic disorders, particularly with symptoms of hyperactivity and irritability. The data are sufficiently encouraging to justify prospective research of this question.