122 research outputs found
Single-locus enrichment without amplification for sequencing and direct detection of epigenetic modifications.
It Takes Two to Tango: Defining an Essential Second Active Site in Pyridoxal 5β²-Phosphate Synthase
The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2) that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5β²-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5β²-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery
Beyond directed evolution: Darwinian selection as a tool for synthetic biology
Synthetic biology is an engineering approach that seeks to design and construct new biological parts, devices and systems, as well as to re-design existing components. However, rationally designed synthetic circuits may not work as expected due to the context-dependence of biological parts. Darwinian selection, the main mechanism through which evolution works, is a major force in creating biodiversity and may be a powerful tool for synthetic biology. This article reviews selection-based techniques and proposes strict Darwinian selection as an alternative approach for the identification and characterization of parts. Additionally, a strategy for fine-tuning of relatively complex circuits by coupling them to a master standard circuit is discussed
Age-related delay in information accrual for faces: Evidence from a parametric, single-trial EEG approach
Background: In this study, we quantified age-related changes in the time-course of face processing
by means of an innovative single-trial ERP approach. Unlike analyses used in previous studies, our
approach does not rely on peak measurements and can provide a more sensitive measure of
processing delays. Young and old adults (mean ages 22 and 70 years) performed a non-speeded
discrimination task between two faces. The phase spectrum of these faces was manipulated
parametrically to create pictures that ranged between pure noise (0% phase information) and the
undistorted signal (100% phase information), with five intermediate steps.
Results: Behavioural 75% correct thresholds were on average lower, and maximum accuracy was
higher, in younger than older observers. ERPs from each subject were entered into a single-trial
general linear regression model to identify variations in neural activity statistically associated with
changes in image structure. The earliest age-related ERP differences occurred in the time window
of the N170. Older observers had a significantly stronger N170 in response to noise, but this age
difference decreased with increasing phase information. Overall, manipulating image phase
information had a greater effect on ERPs from younger observers, which was quantified using a
hierarchical modelling approach. Importantly, visual activity was modulated by the same stimulus
parameters in younger and older subjects. The fit of the model, indexed by R2, was computed at
multiple post-stimulus time points. The time-course of the R2 function showed a significantly slower
processing in older observers starting around 120 ms after stimulus onset. This age-related delay
increased over time to reach a maximum around 190 ms, at which latency younger observers had
around 50 ms time lead over older observers.
Conclusion: Using a component-free ERP analysis that provides a precise timing of the visual
system sensitivity to image structure, the current study demonstrates that older observers
accumulate face information more slowly than younger subjects. Additionally, the N170 appears to
be less face-sensitive in older observers
An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors
Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-Ξ³ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-Ξ³) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-Ξ³ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication
A Cell-Free Microtiter Plate Screen for Improved [FeFe] Hydrogenases
, a potential renewable fuel. Attempts to exploit these catalysts in engineered systems have been hindered by the biotechnologically inconvenient properties of the natural enzymes, including their extreme oxygen sensitivity. Directed evolution has been used to improve the characteristics of a range of natural catalysts, but has been largely unsuccessful for [FeFe] hydrogenases because of a lack of convenient screening platforms. [FeFe] hydrogenase HydA1 with a specific activity βΌ4 times that of the wild-type enzyme. cell extracts, which allows unhindered access to the protein maturation and assay environment
Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases
BACKGROUND: [FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library. CONCLUSIONS/SIGNIFICANCE: [FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy
A competitive integration model of exogenous and endogenous eye movements
We present a model of the eye movement system in which the programming of an eye movement is the result of the competitive integration of information in the superior colliculi (SC). This brain area receives input from occipital cortex, the frontal eye fields, and the dorsolateral prefrontal cortex, on the basis of which it computes the location of the next saccadic target. Two critical assumptions in the model are that cortical inputs are not only excitatory, but can also inhibit saccades to specific locations, and that the SC continue to influence the trajectory of a saccade while it is being executed. With these assumptions, we account for many neurophysiological and behavioral findings from eye movement research. Interactions within the saccade map are shown to account for effects of distractors on saccadic reaction time (SRT) and saccade trajectory, including the global effect and oculomotor capture. In addition, the model accounts for express saccades, the gap effect, saccadic reaction times for antisaccades, and recorded responses from neurons in the SC and frontal eye fields in these tasks. Β© The Author(s) 2010
Integration of Sensory and Reward Information during Perceptual Decision-Making in Lateral Intraparietal Cortex (LIP) of the Macaque Monkey
Single neurons in cortical area LIP are known to carry information relevant to both sensory and value-based decisions that are reported by eye movements. It is not known, however, how sensory and value information are combined in LIP when individual decisions must be based on a combination of these variables. To investigate this issue, we conducted behavioral and electrophysiological experiments in rhesus monkeys during performance of a two-alternative, forced-choice discrimination of motion direction (sensory component). Monkeys reported each decision by making an eye movement to one of two visual targets associated with the two possible directions of motion. We introduced choice biases to the monkeys' decision process (value component) by randomly interleaving balanced reward conditions (equal reward value for the two choices) with unbalanced conditions (one alternative worth twice as much as the other). The monkeys' behavior, as well as that of most LIP neurons, reflected the influence of all relevant variables: the strength of the sensory information, the value of the target in the neuron's response field, and the value of the target outside the response field. Overall, detailed analysis and computer simulation reveal that our data are consistent with a two-stage drift diffusion model proposed by Diederich and Bussmeyer [1] for the effect of payoffs in the context of sensory discrimination tasks. Initial processing of payoff information strongly influences the starting point for the accumulation of sensory evidence, while exerting little if any effect on the rate of accumulation of sensory evidence
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