Molecular insights on the evolution of the lateral and head lantern luciferases and bioluminescence colors in Mastinocerini railroad-worms (Coleoptera: Phengodidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases

The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR) and red-emitting (PxRE) luciferases of railroad worms Phrixotrix viviani and P. hirtus[OLE1]. These luciferases were expressed and purified, and their active-site properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics. The substrate affinities and catalytic efficiency of PxRE luciferase are also higher than those of PxGR luciferase. Fluorescence studies with 8-anilino-1-naphthalene sulfonic acid and 6-p-toluidino-2-naphthalene sulfonic acid showed that the PxRE luciferase luciferin-binding site is more polar than that of PxGR luciferase, and it is sensitive to guanidine. Alutagenesis and modelling studies suggest that several invariant residues in the putative luciferin-binding site of PxRE luciferase cannot interact with excited oxyluciferin. These results suggest that one portion of the luciferin-binding site of the red-emitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.

The influence of the region between residues 220 and 344 and beyond in Phrixotrix railroad worm luciferases green and red bioluminescence

To find the regions having a major influence on the bioluminescence spectra of railroad worm luciferases, we constructed new chimeric luciferases switching the fragments from residues 1-219 and from 220-545 between Phrixotrix viviani (PxvGR; λmax = 548 nm) green light-emitting luciferase and Phrixothrix hirtus (PxhRE; λmax = 623 nm) red light-emitting luciferases. The emission spectrum (λmax = 571 nm) and KM for luciferin in the chimera PxRE220GR (1-219, PxhRE; 220-545, PxvGR) suggested that the region above residue 220 of PxvGR had a major effect on the active site. However, switching the sequence between the residues 226-344 from PxvGR luciferase into PxhRE (PxREGRRE) luciferase resulted in red light emission (λmax = 603 nm), indicating that the region 220-344 by itself does not determine the emission spectrum. Furthermore, the sequence before residue 220 of the green-emitting luciferase is incompatible for light emission with the sequence above residue 220 of PxhRE. These results suggest that the fragments before and after residue 220, which correspond to distinct subdomains, may fold differently in the green- and red-emitting luciferases, affecting the active site conformation.

Few substitutions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site-directed mutagenesis survey

Phrixotrix (railroad worm) luciferases produce bioluminescence in the green and red regions of the spectrum, depending on the location of the lanterns, and are the only luciferases naturally producing red bioluminescence. Comparison of the luciferase sequences showed a set of substitutions that could be involved in bioluminescence colour determination: (a) unique substitutions in the red luciferase replacing otherwise invariant residues; (b) conserved basic residues in the green-yellow emitting luciferases; and (c) an additional R353 residue in red-emitting luciferase (Viviani et al., 1999). To investigate whether these sites have a functional role in bioluminescence colour determination, we performed a site-directed mutagenesis. Natural substitutions in the region 220-344 and residues in the putative luciferin-binding site were also investigated. With the exception of the previously identified substitution of R215 and T226 (Viviani et al., 2002), which display dramatic red-shift effects on the spectrum of green-yellow-emitting luciferases, only a few substitutions had a moderate effect on the spectrum of the green-emitting luciferase. In contrast, no single substitution affected the spectrum of the red-emitting luciferase. The results suggest that the identity of the active site residues is not so critical for determining red bioluminescence in PxRE luciferase. Rather, the conformation assumed during the emitting step could be critical to set up proper interactions with excited oxyluciferin. Copyright ©2007 John Wiley & Sons, Ltd.

Bayesian methodology incorporating expert judgment for ranking countermeasure effectiveness under uncertainty: Example applied to at grade railroad crossings in Korea

This paper describes the formalization and application of a methodology to evaluate the safety benefit of countermeasures in the face of uncertainty. To illustrate the methodology, 18 countermeasures for improving safety of at grade railroad crossings (AGRXs) in the Republic of Korea are considered. Akin to “stated preference” methods in travel survey research, the methodology applies random selection and laws of large numbers to derive accident modification factor (AMF) densities from expert opinions. In a full Bayesian analysis framework, the collective opinions in the form of AMF densities (data likelihood) are combined with prior knowledge (AMF density priors) for the 18 countermeasures to obtain ‘best’ estimates of AMFs (AMF posterior credible intervals). The countermeasures are then compared and recommended based on the largest safety returns with minimum risk (uncertainty). To the author's knowledge the complete methodology is new and has not previously been applied or reported in the literature. The results demonstrate that the methodology is able to discern anticipated safety benefit differences across candidate countermeasures. For the 18 at grade railroad crossings considered in this analysis, it was found that the top three performing countermeasures for reducing crashes are in-vehicle warning systems, obstacle detection systems, and constant warning time systems.

Greener and leaner : unleashing capacity of railroad concrete ties via limit states concept

New knowledge has raised a concern about the cost-ineffective design methods and the true performance of railroad prestressed concrete ties. Because of previous knowledge deficiencies, railway civil and track engineers have been aware of the conservative design methods for structural components in any railway track that rely on allowable stresses and material strength reductions. In particular, railway sleeper (or railroad tie) is an important component of railway tracks and is commonly made of prestressed concrete. The existing code for designing such components makes use of the permissible stress design concept, whereas the fiber stresses over cross sections at initial and final stages are limited by some empirical values. It is believed that the concrete ties complying with the permissible stress concept possess unduly untapped fracture toughness, based on a number of proven experiments and field data. Collaborative research run by the Australian Cooperative Research Centre for Railway Engineering and Technologies (Rail CRC) was initiated to ascertain the reserved capacity of Australian railway prestressed concrete ties that were designed using the existing design code. The findings have led to the development of a new limit-states design concept. This paper highlights the conventional and the new limit-states design philosophies and their implication to both the railway community and the public. © 2011 American Society of Civil Engineers.

Service condition of railroad corridors around the insulated rail joints

Railroad corridors contain large number of Insulated Rail Joints (IRJs) that act as safety critical elements in the circuitries of the signaling and broken rail identification systems. IRJs are regarded as sources of excitation for the passage of loaded wheels leading to high impact forces; these forces in turn cause dips, cross levels and twists to the railroad geometry in close proximity to the sections containing the IRJs in addition to the local damages to the railhead of the IRJs. Therefore, a systematic monitoring of the IRJs in railroad is prudent to mitigate potential risk of their sudden failure (e.g., broken tie plates) under the traffic. This paper presents a simple method of periodic recording of images using time-lapse photography and total station surveying measurements to understand the ongoing deterioration of the IRJs and their surroundings. Over a 500 day period, data were collected to examine the trends in narrowing of the joint gap due to plastic deformation the railhead edges and the dips, cross levels and twists caused to the railroad geometry due to the settlement of ties (sleepers) around the IRJs. The results reflect that the average progressive settlement beneath the IRJs is larger than that under the continuously welded rail, which leads to excessive deviation of railroad profile, cross levels and twists.

A can of worms in a hornets' nest : controversial issues on alcohol and pregnancy

Since the identification of the Foetal Alcohol Syndrome in 1973, researchers have been attempting to discover how much alcohol is deleterious to the developing foetus. This article examines the research evidence of more than a decade suggesting that alcohol is teratogenic. The heated debate between experts in the field both in U.K and Australia is discussed. Implications and recommendations are included.

Studio portrait of Friedel and Johanna Fuld; Worms Portraits Children

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Studio portrait of the grandfather of Friedel and Johanna Fuld; Worms Portraits Men

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Studio portrait of the grandmother of Friedel and Johanna Fuld; Worms Portraits Women

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Destroyed Railroad Wagon in Mero Laburcz Military; World War I

Bernhard Bardach World War I Album I

Genealogie der Familie Eberstadt aus Worms 1654-

Three volumes of genealogy and family trees of the Eberstadt family.