Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

Modeling of optical detection of spin-polarized carrier injection into light-emitting devices

We investigate the emission of multimodal polarized light from light emitting devices due to spin-aligned carrier injection. The results are derived through operator Langevin equations, which include thermal and carrier-injection fluctuations, as well as nonradiative recombination and electronic g-factor temperature dependence. We study the dynamics of the optoelectronic processes and show how the temperature-dependent g factor and magnetic field affect the degree of polarization of the emitted light. In addition, at high temperatures, thermal fluctuation reduces the efficiency of the optoelectronic detection method for measuring the degree of spin polarization of carrier injection into nonmagnetic semicondutors.

Snakes and cats in the flower bed: Fast detection is not specific to pictures of fear-relevant animals

The observation that snakes and spiders are found faster among flowers and mushrooms than vice versa and that this search advantage is independent of set size supports the notion that fear-relevant stimuli are processed preferentially in a dedicated fear module. Experiment I replicated the faster identification of snakes and spiders but also found a set size effect in a blocked, but not in a mixed-trial, sequence. Experiment 2 failed to find faster identification of snake and spider deviants relative to other animals among flowers and mushrooms and provided evidence for a search advantage for pictures of animals, irrespective of their fear relevance. These findings suggest that results from the present visual search task cannot support the notion of preferential processing of fear relevance.

Collagen in the scarless fetal skin wound: Detection with Picrosirius-polarization

Our group has developed an ovine model of deep dermal, partial-thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross-linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a response to burn injury.

Development of an oligonucleotide-based SNP detection method on lateral flow strips using hexapet tages

Detection of point mutations or single nucleotide polymorphisms (SNPs) is important in relation to disease susceptibility or detection in pathogens of mutations determining drug resistance or host range. There is an emergent need for rapid detection methods amenable to point-of-care applications. The purpose of this study was to reduce to practice a novel method for SNP detection and to demonstrate that this technology can be used downstream of nucleic acid amplification. The authors used a model system to develop an oligonucleotide-based SNP detection system on nitrocellulose lateral flow strips. To optimize the assay they used cloned sequences of the herpes simplex virus-1 (HSV-1) DNA polymerase gene into which they introduced a point mutation. The assay system uses chimeric polymerase chain reaction (PCR) primers that incorporate hexameric repeat tags ("hexapet tags"). The chimeric sequences allow capture of amplified products to predefined positions on a lateral flow strip. These "hexapet" sequences have minimal cross-reactivity and allow specific hybridization-based capture of the PCR products at room temperature onto lateral flow strips that have been striped with complementary hexapet tags. The allele-specific amplification was carried out with both mutant and wild-type primer sets present in the PCR mix ("competitive" format). The resulting PCR products carried a hexapet tag that corresponded with either a wild-type or mutant sequence. The lateral flow strips are dropped into the PCR reaction tube, and mutant sequence and wild-type sequences diffuse along the strip and are captured at the corresponding position on the strip. A red line indicative of a positive reaction is visible after 1 minute. Unlike other systems that require separate reactions and strips for each target sequence, this system allows multiplex PCR reactions and multiplex detection on a single strip or other suitable substrates. Unambiguous visual discrimination of a point mutation under room temperature hybridization conditions was achieved with this model system in 10 minutes after PCR. The authors have developed a capture-based hybridization method for the detection and discrimination of HSV-1 DNA polymerase genes that contain a single nucleotide change. It has been demonstrated that the hexapet oligonucleotides can be adapted for hybridization on the lateral flow strip platform for discrimination of SNPs. This is the first step in demonstrating SNP detection on lateral flow using the hexapet oligonucleotide capture system. It is anticipated that this novel system can be widely used in point-of-care settings.

Conventional detection methodology is limiting our ability to understand the roles and functions of fine roots

We lack a thorough conceptual and functional understanding of fine roots. Studies that have focused on estimating the quantity of fine roots provide evidence that they dominate overall plant root length. We need a standard procedure to quantify root length/biomass that takes proper account of fine roots. Here we investigated the extent to which root length/biomass may be underestimated using conventional methodology, and examined the technical reasons that could explain such underestimation. Our discussion is based on original X-ray-based measurements and on a literature review spanning more than six decades. We present evidence that root-length recovery depends strongly on the observation scale/spatial resolution at which measurements are carried out; and that observation scales/resolutions adequate for fine root detection have an adverse impact on the processing times required to obtain precise estimates. We conclude that fine roots are the major component of root systems of most (if not all) annual and perennial plants. Hence plant root systems could be much longer, and probably include more biomass, than is widely accepted.

Statistics of single-electron signals in electron-multiplying charge-coupled devices

Electron-multiplying charge coupled devices promise to revolutionize ultrasensitive optical imaging. The authors present a simple methodology allowing reliable measurement of camera characteristics and statistics of single-electron events, compare the measurements to a simple theoretical model, and report camera performance in a truly photon-counting regime that eliminates the excess noise related to fluctuations of the multiplication gain.

Spin-detection in a quantum electromechanical shuttle system

We study the electrical transport of a harmonically bound, single-molecule C-60 shuttle operating in the Coulomb blockade regime, i.e. single electron shuttling. In particular, we examine the dependance of the tunnel current on an ultra-small stationary force exerted on the shuttle. As an example, we consider the force exerted on an endohedral N@C-60 by the magnetic field gradient generated by a nearby nanomagnet. We derive a Hamiltonian for the full shuttle system which includes the metallic contacts, the spatially dependent tunnel couplings to the shuttle, the electronic and motional degrees of freedom of the shuttle itself and a coupling of the shuttle's motion to a phonon bath. We analyse the resulting quantum master equation and find that, due to the exponential dependence of the tunnel probability on the shuttle-contact separation, the current is highly sensitive to very small forces. In particular, we predict that the spin state of the endohedral electrons of N@C-60 in a large magnetic gradient field can be distinguished from the resulting current signals within a few tens of nanoseconds. This effect could prove useful for the detection of the endohedral spin-state of individual paramagnetic molecules such as N@C-60 and P@C-60, or the detection of very small static forces acting on a C-60 shuttle.

An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients

We describe the development of a capture enzyme-linked immunosorbent assay for the detection of the dengue virus nonstructural protein NS1. The assay employs rabbit polyclonal and monoclonal antibodies as the capture and detection antibodies, respectively. Immunoaffinity-purified NS1 derived from dengue 2 virus-infected cells was used as a standard to establish a detection sensitivity of approximately 4 ng/ml for an assay employing monoclonal antibodies recognizing a dengue 2 serotype-specific epitope. A number of serotype cross-reactive monoclonal antibodies were also shown to be suitable probes for the detection of NS1 expressed by the remaining three dengue virus serotypes. Examination of clinical samples demonstrated that the assay was able to detect NS1 with minimal interference from serum components at the test dilutions routinely used, suggesting that it could form the basis of a useful additional diagnostic test for dengue virus infection. Furthermore, quantitation of NS1 levels in patient sera may prove to be a valuable surrogate marker for viremia. Surprisingly high levels of NS1, as much as 15 mu g/ml, were found in acute-phase sera taken hom some of the patients experiencing serologically confirmed dengue 2 virus secondary infections but was not detected in the convalescent sera of these patients. In contrast, NS1 could not be detected in either acute-phase or convalescent serum samples taken from patients with serologically confirmed primary infection. The presence of high levels of secreted NS1 in the sera of patients experiencing secondary dengue virus infections, and in the context of an anamnestic antibody response, suggests that NS1 may contribute significantly to the formation of the circulating immune complexes that are suspected to play an important role in the pathogenesis of severe dengue disease.