Molecular and biochemical basis for the loss of bioluminescence in the dinoflagellate Noctiluca scintillans along the west coast of the USA.

The globally distributed heterotrophic dinoflagellate Noctiluca scintillans (Macartney) Kofoid & Swezy is well known for its dense blooms and prominent displays of bioluminescence. Intriguingly, along the west coast of the USA its blooms are not bioluminescent. We investigated the basis for the regional loss of bioluminescence using molecular, cellular and biochemical analyses of isolates from different geographic regions. Prominent differences of the non-bioluminescent strains were: (1) the fused luciferase and luciferin binding protein gene (lcf/lbp) was present but its transcripts were undetectable; (2) lcf/lbp contained multiple potentially deleterious mutations; (3) the substrate luciferin was absent, based on the lack of luciferin blue autofluorescence and the absence of luciferin derived metabolites; (4) although the cells possessed scintillons, the vesicles that contain the luminescent chemistry, electron microscopy revealed additional scintillon-like vesicles with an atypical internal structure; (5) cells isolated from the California coast were 43% smaller in size than bioluminescent cells from the Gulf of Mexico. Phylogenetic analyses based on the large subunit of rDNA did not show divergence of the non-bioluminescent population in relation to other bioluminescent N. scintillans from the Pacific Ocean and Arabian Sea. Our study demonstrates that gene silencing and the lack of the luciferin substrate have resulted in the loss of a significant dinoflagellate functional trait over large spatial scales in the ocean. As the bioluminescence system of dinoflagellates is well characterized, non-bioluminescent N. scintillans is an ideal model to explore the evolutionary and ecological mechanisms that lead to intraspecific functional divergence in natural dinoflagellate populations.

Rational engineering of photosynthetic electron flux enhances light-powered cytochrome P450 activity.

In this study, we exploited a modified photosynthetic electron transfer chain (PET) in the model cyanobacterium Synechococcus PCC 7002, where electrons derived from water-splitting are used to power reactions catalyzed by a heterologous cytochrome P450 (CYP1A1). A simple in vivo fluorescent assay for CYP1A1 activity was employed to determine the impact of rationally engineering of photosynthetic electron flow. This showed that knocking out a subunit of the type I NADH dehydrogenase complex (NDH-1), suggested to be involved in cyclic photosynthetic electron flow (ΔndhD2), can double the activity of CYP1A1, with a concomitant increase in the flux of electrons from photosynthesis. This also resulted in an increase in cellular adenosine triphosphate (ATP) and the ATP/nicotinamide adenine dinucleotide phosphate (NADPH) ratio, suggesting that expression of a heterologous electron sink in photosynthetic organisms can be used to modify the bioenergetic landscape of the cell. We therefore demonstrate that CYP1A1 is limited by electron supply and that photosynthesis can be re-engineered to increase heterologous P450 activity for the production of high-value bioproducts. The increase in cellular ATP achieved could be harnessed to support metabolically demanding heterologous processes. Furthermore, this experimental system could provide valuable insights into the mechanisms of photosynthesis.

Cloning of a copper resistance gene cluster from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery.

A copper resistance gene cluster (6 genes, ∼8.2 kb) was isolated from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery (RR). Following integration of a narrow-host-range plasmid vector adjacent to the target region in the Synechocystis genome (pSYSX), DNA was isolated from transformed cells and the plasmid plus flanking sequence circularized by recombineering to precisely clone the gene cluster. Complementation of a copper-sensitive Escherichia coli mutant demonstrated the functionality of the pcopM gene encoding a copper-binding protein. RR provides a novel alternative method for cloning large DNA fragments from species that can be transformed by homologous recombination.

Fluorescent protein-mediated colour polymorphism in reef corals: multicopy genes extend the adaptation/acclimatization potential to variable light environments.

The genomic framework that enables corals to adjust to unfavourable conditions is crucial for coral reef survival in a rapidly changing climate. We have explored the striking intraspecific variability in the expression of coral pigments from the green fluorescent protein (GFP) family to elucidate the genomic basis for the plasticity of stress responses among reef corals. We show that multicopy genes can greatly increase the dynamic range over which corals can modulate transcript levels in response to the light environment. Using the red fluorescent protein amilFP597 in the coral Acropora millepora as a model, we demonstrate that its expression increases with light intensity, but both the minimal and maximal gene transcript levels vary markedly among colour morphs. The pigment concentration in the tissue of different morphs is strongly correlated with the number of gene copies with a particular promoter type. These findings indicate that colour polymorphism in reef corals can be caused by the environmentally regulated expression of multicopy genes. High-level expression of amilFP597 is correlated with reduced photodamage of zooxanthellae under acute light stress, supporting a photoprotective function of this pigment. The cluster of light-regulated pigment genes can enable corals to invest either in expensive high-level pigmentation, offering benefits under light stress, or to rely on low tissue pigment concentrations and use the conserved resources for other purposes, which is preferable in less light-exposed environments. The genomic framework described here allows corals to pursue different strategies to succeed in habitats with highly variable light stress levels. In summary, our results suggest that the intraspecific plasticity of reef corals' stress responses is larger than previously thought.

Bayesian sample size calculation for estimation of the difference between two binomial proportions.

In this study, we discuss a decision theoretic or fully Bayesian approach to the sample size question in clinical trials with binary responses. Data are assumed to come from two binomial distributions. A Dirichlet distribution is assumed to describe prior knowledge of the two success probabilities p1 and p2. The parameter of interest is p = p1 - p2. The optimal size of the trial is obtained by maximising the expected net benefit function. The methodology presented in this article extends previous work by the assumption of dependent prior distributions for p1 and p2.

The pea SAD short-chain dehydrogenase/reductase: quinone reduction, tissue distribution, and heterologous expression.

The pea (Pisum sativum) tetrameric short-chain alcohol dehydrogenase-like protein (SAD) family consists of at least three highly similar members (SAD-A, -B, and -C). According to mRNA data, environmental stimuli induce SAD expression. The aim of this study was to characterize the SAD proteins by examining their catalytic function, distribution in pea, and induction in different tissues. In enzyme activity assays using a range of potential substrates, the SAD-C enzyme was shown to reduce one- or two-ring-membered quinones lacking long hydrophobic hydrocarbon tails. Immunological assays using a specific antiserum against the protein demonstrated that different tissues and cell types contain small amounts of SAD protein that was predominantly located within epidermal or subepidermal cells and around vascular tissue. Particularly high local concentrations were observed in the protoderm of the seed cotyledonary axis. Two bow-shaped rows of cells in the ovary and the placental surface facing the ovule also exhibited considerable SAD staining. Ultraviolet-B irradiation led to increased staining in epidermal and subepidermal cells of leaves and stems. The different localization patterns of SAD suggest functions both in development and in responses to environmental stimuli. Finally, the pea SAD-C promoter was shown to confer heterologous wound-induced expression in Arabidopsis (Arabidopsis thaliana), which confirmed that the inducibility of its expression is regulated at the transcriptional level.

A behavioural Bayes approach to the determination of sample size for clinical trials considering efficacy and safety: imbalanced sample size in treatment groups.

The behavioural Bayes approach to sample size determination for clinical trials assumes that the number of subsequent patients switching to a new drug from the current drug depends on the strength of the evidence for efficacy and safety that was observed in the clinical trials. The optimal sample size is the one which maximises the expected net benefit of the trial. The approach has been developed in a series of papers by Pezeshk and the present authors (Gittins JC, Pezeshk H. A behavioral Bayes method for determining the size of a clinical trial. Drug Information Journal 2000; 34: 355-63; Gittins JC, Pezeshk H. How Large should a clinical trial be? The Statistician 2000; 49(2): 177-87; Gittins JC, Pezeshk H. A decision theoretic approach to sample size determination in clinical trials. Journal of Biopharmaceutical Statistics 2002; 12(4): 535-51; Gittins JC, Pezeshk H. A fully Bayesian approach to calculating sample sizes for clinical trials with binary responses. Drug Information Journal 2002; 36: 143-50; Kikuchi T, Pezeshk H, Gittins J. A Bayesian cost-benefit approach to the determination of sample size in clinical trials. Statistics in Medicine 2008; 27(1): 68-82; Kikuchi T, Gittins J. A behavioral Bayes method to determine the sample size of a clinical trial considering efficacy and safety. Statistics in Medicine 2009; 28(18): 2293-306; Kikuchi T, Gittins J. A Bayesian procedure for cost-benefit evaluation of a new drug in multi-national clinical trials. Statistics in Medicine 2009 (Submitted)). The purpose of this article is to provide a rationale for experimental designs which allocate more patients to the new treatment than to the control group. The model uses a logistic weight function, including an interaction term linking efficacy and safety, which determines the number of patients choosing the new drug, and hence the resulting benefit. A Monte Carlo simulation is employed for the calculation. Having a larger group of patients on the new drug in general makes it easier to recruit patients to the trial and may also be ethically desirable. Our results show that this can be done with very little if any reduction in expected net benefit.

The leicester Doppler phantom--a digital electronic phantom for ultrasound pulsed Doppler system testing.

Doppler flow and string phantoms have been used to assess the performance of ultrasound Doppler systems in terms of parameters such as sensitivity, velocity accuracy and sample volume registration. However, because of the nature of their construction, they cannot challenge the accuracy and repeatability of modern digital ultrasound systems or give objective measures of system performance. Electronic Doppler phantoms are able to make use of electronically generated test signals, which may be controlled precisely in terms of frequency, amplitude and timing. The Leicester Electronic Doppler Phantom uses modern digital signal processing methods and field programmable gate array technology to overcome some of the limitations of previously described electronic phantoms. In its present form, it is able to give quantitative graphical assessments of frequency response and range gate characteristics, as well as measures of dynamic range and velocity measurement accuracy. The use of direct acoustic coupling eliminates uncertainties caused by Doppler beam effects, such as intrinsic spectral broadening, but prevents their evaluation.

A behavioral Bayes method to determine the sample size of a clinical trial considering efficacy and safety.

It is necessary for the calculation of sample size to achieve the best balance between the cost of a clinical trial and the possible benefits from a new treatment. Gittins and Pezeshk developed an innovative (behavioral Bayes) approach, which assumes that the number of users is an increasing function of the difference in performance between the new treatment and the standard treatment. The better a new treatment, the more the number of patients who want to switch to it. The optimal sample size is calculated in this framework. This BeBay approach takes account of three decision-makers, a pharmaceutical company, the health authority and medical advisers. Kikuchi, Pezeshk and Gittins generalized this approach by introducing a logistic benefit function, and by extending to the more usual unpaired case, and with unknown variance. The expected net benefit in this model is based on the efficacy of the new drug but does not take account of the incidence of adverse reactions. The present paper extends the model to include the costs of treating adverse reactions and focuses on societal cost-effectiveness as the criterion for determining sample size. The main application is likely to be to phase III clinical trials, for which the primary outcome is to compare the costs and benefits of a new drug with a standard drug in relation to national health-care.

The choice of sample size: a mixed Bayesian / frequentist approach.

Sample size computations are largely based on frequentist or classical methods. In the Bayesian approach the prior information on the unknown parameters is taken into account. In this work we consider a fully Bayesian approach to the sample size determination problem which was introduced by Grundy et al. and developed by Lindley. This approach treats the problem as a decision problem and employs a utility function to find the optimal sample size of a trial. Furthermore, we assume that a regulatory authority, which is deciding on whether or not to grant a licence to a new treatment, uses a frequentist approach. We then find the optimal sample size for the trial by maximising the expected net benefit, which is the expected benefit of subsequent use of the new treatment minus the cost of the trial.

Doppler ultrasound detection of side-vessel occlusion: an in vitro study.

BACKGROUND AND PURPOSE: Small-vessel knock is a recently reported Doppler ultrasound finding that has been identified in patients with cerebral ischemia. It has been hypothesized that knock-type signals are linked to the presence of either small-vessel occlusion or wall motion. The aim of this study was to investigate the origins of "knock-type" signals by reproducing occlusion of a peripheral artery model in vitro. METHODS: Synthetic bifurcations were fabricated from glass and latex and placed in a flow-rig mimicking physiological blood-flow conditions. The glass model permitted study of fluid flow in the absence of wall motion, whereas the latex model also produced wall motion effects. Vessels were artificially obstructed to examine Doppler signal characteristics associated with blood flow and wall motion. RESULTS: Complete obstruction of the peripheral branch of the glass model revealed discrete (<100 ms) knock-type signals caused by local fluid flow in the occluded branch. Imaging of the obstructed vessel using color Doppler revealed forward and reflected flow. The walls produced periodic bidirectional knock-type signals, which occurred during systole and were not related to the presence of an obstruction. CONCLUSIONS: In our laboratory model, transcranial Doppler ultrasound was found to be capable of detecting knock signals produced by circulating fluid within an occluded branch. However, because similar signals are also generated by nonpathological wall motion, these results cannot be directly translated to a clinical setting. Clinicians should be careful to avoid casual overinterpretation of transcranial Doppler ultrasound data.

Phytoplankton calcification in a high-CO2 world.

Ocean acidification in response to rising atmospheric CO2 partial pressures is widely expected to reduce calcification by marine organisms. From the mid-Mesozoic, coccolithophores have been major calcium carbonate producers in the world's oceans, today accounting for about a third of the total marine CaCO3 production. Here, we present laboratory evidence that calcification and net primary production in the coccolithophore species Emiliania huxleyi are significantly increased by high CO2 partial pressures. Field evidence from the deep ocean is consistent with these laboratory conclusions, indicating that over the past 220 years there has been a 40% increase in average coccolith mass. Our findings show that coccolithophores are already responding and will probably continue to respond to rising atmospheric CO2 partial pressures, which has important implications for biogeochemical modeling of future oceans and climate.

Improving performance of pulse compression in a Doppler ultrasound system using amplitude modulated chirps and Wiener filtering.

The use of coded excitation and pulse compression in transcranial Doppler (TCD) ultrasound systems is in the very early stages; the optimal processing scheme has yet to be determined. This study uses linear frequency-modulated (FM) chirps with 0.8-MHz bandwidth and compares the use of pulses with and without amplitude modulation and also matched filtering vs. Wiener filtering. The results demonstrate that using amplitude-modulated pulses vastly improves the axial resolution and provides a more predictable mainlobe-to-sidelobe distance in the radiofrequency (RF) signal. It is also shown that a Wiener filter can provide better performance than a matched filter, in terms of axial resolution and sidelobe level in the RF signal and signal-to-noise ratio in conventional sonograms. Although this study uses a TCD system, the techniques described are equally as applicable in other Doppler ultrasound devices.

A Bayesian cost-benefit approach to the determination of sample size in clinical trials.

Current practice for sample size computations in clinical trials is largely based on frequentist or classical methods. These methods have the drawback of requiring a point estimate of the variance of the treatment effect and are based on arbitrary settings of type I and II errors. They also do not directly address the question of achieving the best balance between the cost of the trial and the possible benefits from using the new treatment, and fail to consider the important fact that the number of users depends on the evidence for improvement compared with the current treatment. Our approach, Behavioural Bayes (or BeBay for short), assumes that the number of patients switching to the new medical treatment depends on the strength of the evidence that is provided by clinical trials, and takes a value between zero and the number of potential patients. The better a new treatment, the more the number of patients who want to switch to it and the more the benefit is obtained. We define the optimal sample size to be the sample size that maximizes the expected net benefit resulting from a clinical trial. Gittins and Pezeshk (Drug Inf. Control 2000; 34:355-363; The Statistician 2000; 49(2):177-187) used a simple form of benefit function and assumed paired comparisons between two medical treatments and that the variance of the treatment effect is known. We generalize this setting, by introducing a logistic benefit function, and by extending the more usual unpaired case, without assuming the variance to be known.

Coded excitation in TCD ultrasound systems to improve axial resolution.

The radiofrequency (RF) signal from a transcranial Doppler (TCD) ultrasound system allows us to track the motion of an embolus in a 2-D space of time and depth. The technique is limited by the narrow bandwidth (long duration) of the transmitted pulse because this provides poor axial resolution. This study aimed to assess whether implementing coded excitation and pulse compression in a TCD system would be a feasible means of increasing the bandwidth and hence improving the axial resolution, without the need for reducing the pulse length and increasing peak power levels. Embolic signals were collected in vitro from a flow phantom using a TCD system, which alternately transmitted coded and noncoded pulses. This allowed the same event to be investigated using the two different types of processing. Quantitative and qualitative measures were used to compare the two processing methods. The in vitro results were promising. They showed that the axial resolution could be improved, on average, by a factor of 6.6, using a pulse length of 13 micros and a chirp bandwidth of 0.8 MHz. This was reduced to a factor of 6 when a temporal bone sample was placed between the transducer and the flow phantom. Qualitatively the journey of an embolus was easier to track using the pulse compressed signal. Sonograms could be generated from smaller receive gates using the pulse compressed signal while still achieving an adequate signal-to-noise ratio. We found that it is both feasible and beneficial to implement coded excitation and pulse compression in a TCD system.

Bayesian approach to determine the number of subsequent users of a new treatment.

The aim of this article is to discuss the distribution function of the number of subsequent users of a new treatment. A Bayesian approach is applied. Using the fact that the number of subsequent users of the new treatment will not be high, unless it is, in the statistical and also in the clinical sense, significantly better than the existing one, we obtain the distribution function of the number of subsequent users of a new treatment for which we assume the data have come from a normal distribution.

RF signals provide additional information on embolic events recorded during TCD monitoring.

Transcranial Doppler ultrasound (TCD) is commonly used to detect embolic signals in the cerebral circulation. However, current techniques to discriminate between signals from emboli and artifacts are subjective and ambiguous. The radiofrequency (RF) signal provides an extra dimension to the information available from conventional TCD systems that may help to interpret complex events. Artifacts generated by healthy volunteers and embolic signals recorded from a flow phantom were used to characterize the appearance of the two types of event. Characteristics of events, recorded during and immediately after carotid endarterectomy surgery, were compared with those from known sources. Additional information was provided by the RF signal on events recorded during TCD monitoring thus aiding classification. The RF signal may have a role as a "gold standard" for embolus detection.

Limits of uncertainty in measured values of embolus-to-blood ratios in dual-frequency TCD recordings due to nonidentical sample volume shapes.

Transcranial Doppler (TCD) ultrasound (US) is widely used to detect cerebral embolisation. A drawback of the technique is that it is difficult to distinguish between small gaseous emboli and large solid emboli. One potential solution is to make measurements from blood and emboli at two different frequencies, because the frequency-dependence of ultrasonic backscatter will be different for the two types of emboli. Unfortunately, it is not possible to produce two identical sample volumes at two different frequencies, and this will introduce an uncertainty into any such measurements. Experimental measurements have been made of US fields distorted by temporal bone at 2.0 MHz and 2.5 MHz, and a mathematical model has been developed to examine the uncertainty introduced by the different field shapes at these frequencies. The results suggest that the uncertainties are sufficiently large (on the order of 2 to 4 dB) that a significant percentage of emboli are likely to be misclassified by the dual-frequency method.

Transgene expression in the vegetative tissues of apple driven by the vascular-specific rolC and CoYMV promoters.

The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of beta-glucuronidase (GUS) activity by fluorometric assay. The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter. These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter. In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate. This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem. Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem.

Identification of a novel nuclear factor-binding site in the Pisum sativum sad gene promoters.

DNA fragments containing the 5' promoter regions of the Pisum sativum sadA and sadC genes were amplified from genomic DNA, cloned and sequenced. These sequences contain a number of conserved cis-acting elements, which are potentially involved in stress-induced transcription of the sad genes. To determine whether any of the identified elements are active in binding nuclear factors in vitro, 11 60-bp overlapping (by 30 bp) DNA probe fragments covering the proximal sadC promoter sequence (360 bp) were used in electrophoretic mobility shift assays with competition. Binding activities were compared in nuclear extracts from control, UV-B-stressed and wounded pea leaves. The pattern of DNA binding was almost identical with all three extracts, with one 30-bp region being the predominant site for factor binding. Using overlapping sub-fragments of this region, the majority of the specific binding could be attributed to the novel 11-bp GC-rich sequence GTGGCGCCCAC. An almost identical sequence is conserved in the sadA promoter. This motif has features in common with a number of recognised cis-elements, which suggests a possible binding site for factors which play a role in regulating sad gene transcription.