Silicon photomultiplier (SPM) detection of low-level bioluminescence for the development of deployable whole-cell biosensors: possibilities and limitations

Whole-cell bacterial bioreporters await miniaturized photon counting modules with high sensitivity and robust compatible hardware to fulfill their promise of versatile, on-site biosensor functionality. In this study, we explore the photon counting readout properties of the silicon photomultiplier (SPM) with a thermoelectric cooler and the possibilities of detecting low-level bioluminescent signals. Detection performance was evaluated through a simulated LED light source and the bioluminescence produced by the genetically engineered Pseudomonas fluorescens bacterial bioreporter 5RL. Compared with the conventional photomultiplier tube (PMT), the results revealed that the cooled SPM exhibits a wider linear response to inducible substrate concentrations (salicylate) ranging from 250 to 5000 ppb. Although cooling of the SPM lowered dark count rates and improved the minimum detectable signal, and the application of a digital filter enhanced the signal-to-noise ratio, the detection of very low light signals is still limited and remains a challenge in the design of compact photon counting systems.     

Field applications of genetically engineered microorganisms for bioremediation processes

Genetically engineered microorganisms (GEMs) have shown potential for bioremediation applications in soil, groundwater, and activated sludge environments, exhibiting enhanced degradative capabilities encompassing a wide range of chemical contaminants. However, the vast majority of studies pertaining to genetically engineered microbial bioremediation are supported by laboratory-based experimental data. In general, relatively few examples of GEM applications in environmental ecosystems exist. Unfortunately, the only manner in which to fully address the competence of GEMs in bioremediation efforts is through long-term field release studies. It is therefore essential that field studies be performed to acquire the requisite information for determining the overall effectiveness and risks associated with GEM introduction into natural ecosystems.     

Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content

Tocotrienols are the primary form of vitamin E in seeds of most monocot plants, including cereals such as rice and wheat. As potent antioxidants, tocotrienols contribute to the nutritive value of cereal grains in human and livestock diets. cDNAs encoding homogentisic acid geranylgeranyl transferase (HGGT), which catalyzes the committed step of tocotrienol biosynthesis, were isolated from barley, wheat and rice seeds. Transgenic expression of the barley HGGT in Arabidopsis thaliana leaves resulted in accumulation of tocotrienols, which were absent from leaves of nontransformed plants, and a 10- to 15-fold increase in total vitamin E antioxidants (tocotrienols plus tocopherols). Overexpression of the barley HGGT in corn seeds resulted in an increase in tocotrienol and tocopherol content of as much as six-fold. These results provide insight into the genetic basis for tocotrienol biosynthesis in plants and demonstrate the ability to enhance the antioxidant content of crops by introduction of an enzyme that redirects metabolic flux.     

An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi

The hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii, whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles. We have performed an extensive re-annotation of the genome of P. abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology. Many new functions are predicted for both informational and operational proteins. Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features. The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree. However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P. abyssi genome.     

Glucocorticoids inhibit interconversion of 7-hydroxy and 7-oxo metabolites of dehydroepiandrosterone: a role for 11beta-hydroxysteroid dehydrogenases?

The cytochrome p450-dependent formation and subsequent interconversion of dehydroepiandrosterone (DHEA) metabolites 7 alpha-hydroxy-DHEA (7 alpha-OH-DHEA), 7 beta-hydroxy-DHEA (7 beta-OH-DHEA), and 7-oxo-DHEA was observed in human, pig, and rat liver microsomal fractions. Rat liver mitochondria and nuclei also converted DHEA to 7 alpha-OH-DHEA and 7-oxo-DHEA, but at a lower rate. With NADP(+), and less so with NAD(+), rat, pig, and human liver microsomes and rat liver mitochondria and nuclei converted 7 alpha-OH-DHEA to 7-oxo-DHEA. This reaction was inhibited by corticosterone and the 11 beta-hydroxysteroid dehydrogenase (11 betaHSD) inhibitor carbenoxolone (CBX). The conversion of 7 alpha-OH-DHEA to 7-oxo-DHEA by rat kidney occurred at higher rates with NAD(+) than with NADP(+) and was inhibited by corticosterone. With NADPH, 7-oxo-DHEA was converted to unidentified hydroxylated metabolites and low levels of 7 alpha-OH-DHEA by rat liver microsomes. In contrast, pig liver microsomal fractions reduced 7-oxo-DHEA to nearly equal amounts of 7 alpha- and 7 beta-OH-DHEA, while human fractions produced mainly 7 beta-OH-DHEA. Dehydrocorticosterone inhibited the reduction to both isomers by pig liver microsomes, but only to 7 alpha-OH-DHEA by human microsomes; CBX inhibited both reactions. Rat kidney did not reduce 7-oxo-DHEA with either NADPH or NADH. These results demonstrate that DHEA is first converted in liver to 7 alpha-OH-DHEA, which is subsequently oxidized to 7-oxo-DHEA in both liver and kidney. In liver, interconversion of 7-oxo-DHEA and 7-OH-DHEA isomers is largely catalyzed by 11 betaHSD1, while in kidney 11 betaHSD2 (NAD(+)-dependent) and 11 betaHSD3 (NADP(+)-dependent) likely catalyze the unidirectional oxidation of 7 alpha-hydroxy-DHEA to 7-oxo-DHEA. Distinct species-specific routes of metabolism of DHEA and the interconversion of its metabolites obviate extrapolation of animal studies to humans.     

Calcium currents in embryonic and neonatal mammalian skeletal muscle

The whole-cell patch-clamp technique was used to study the properties of inward ionic currents found in primary cultures of rat and mouse skeletal myotubes and in freshly dissociated fibers of the flexor digitorum brevis muscle of rats. In each of these cell types, test depolarizations from the holding potential (-80 or -90 mV) elicited three distinct inward currents: a sodium current (INa) and two calcium currents. INa was the dominant inward current: under physiological conditions, the maximum inward INa was estimated to be at least 30-fold larger than either of the calcium currents. The two calcium currents have been termed Ifast and Islow, corresponding to their relative rates of activation. Ifast was activated by test depolarizations to around -40 mV and above, peaked in 10-20 ms, and decayed to baseline in 50-100 ms. Islow was activated by depolarizations to approximately 0 mV and above, peaked in 50-150 ms, and decayed little during a 200-ms test pulse. Ifast was inactivated by brief, moderate depolarizations; for a 1-s change in holding potential, half-inactivation occurred at -55 to -45 mV and complete inactivation occurred at -40 to -30 mV. Similar changes in holding potential had no effect on Islow. Islow was, however, inactivated by brief, strong depolarizations (e.g., 0 mV for 2 s) or maintained, moderate depolarizations (e.g., -40 mV for 60 s). Substitution of barium for calcium had little effect on the magnitude or time course of either Ifast or Islow. The same substitution shifted the activation curve for Islow approximately 10 mV in the hyperpolarizing direction without affecting the activation of Ifast. At low concentrations (50 microM), cadmium preferentially blocked Islow compared with Ifast, while at high concentrations (1 mM), it blocked both Ifast and Islow completely. The dihydropyridine calcium channel antagonist (+)-PN 200-110 (1 microM) caused a nearly complete block of Islow without affecting Ifast. At a holding potential of -80 mV, the half-maximal blocking concentration (K0.5) for the block of Islow by (+)-PN 200-110 was 182 nM. At depolarized holding potentials that inactivated Islow by 35-65%, K0.5 decreased to 5.5 nM.     

Effect of Cookery and Holding on Hams and Turkey Rolls Contaminated with Clostridium perfringens

Canned hams, turkey rolls, and ground-beef casseroles were inoculated with a mixture of vegetative cells and spores of selected strains of Clostridium perfringens, in approximately known numbers. After cooking and holding at different temperatures for various times, samples of the food were plated directly on sulfadiazine-polymixin-sulfite-agar. In all cases, small but measurable percentages of the organisms survived cookery. The number of cells viable after cookery of the ham or turkey was influenced by the position of the slice of meat in the roast as well as by the final temperature to which the product was heated. Plate counts for turkey or beef casserole held at temperatures in the range of 5 to 10 C for 48 hr indicated stabilization of the population or a tendency to decrease. At 24 C, the multiplication of cells was apparent in 4 hr and rapid in 6 hr. When the food was maintained at 68 C, populations remained viable for 6 hr and the counts did not change markedly. In turkey maintained at 37 C, the number of cells increased sharply within 4 hr.     

Phosphorylation of myosin A regulates gliding motility and is essential for Plasmodium transmission

Malaria‐causing parasites rely on an actin–myosin‐based motor for the invasion of different host cells and tissue traversal in mosquitoes and vertebrates. The unusual myosin A of Plasmodium spp. has a unique N‐terminal extension, which is important for red blood cell invasion by P. falciparum merozoites in vitro and harbors a phosphorylation site at serine 19. Here, using the rodent‐infecting P. berghei we show that phosphorylation of serine 19 increases ookinete but not sporozoite motility and is essential for efficient transmission of Plasmodium by mosquitoes as S19A mutants show defects in mosquito salivary gland entry. S19A along with E6R mutations slow ookinetes and salivary gland sporozoites in both 2D and 3D environments. In contrast to data from purified proteins, both E6R and S19D mutations lower force generation by sporozoites. Our data show that the phosphorylation cycle of S19 influences parasite migration and force generation and is critical for optimal migration of parasites during transmission from and to the mosquito.     

Structural Genomics of Eukaryotic Targets at a Laboratory Scale

Structural genomics programs are distributed worldwide and funded by large institutions such as the NIH in United-States, the RIKEN in Japan or the European Commission through the SPINE network in Europe. Such initiatives, essentially managed by large consortia, led to technology and method developments at the different steps required to produce biological samples compatible with structural studies. Besides specific applications, method developments resulted mainly upon miniaturization and parallelization. The challenge that academic laboratories faces to pursue structural genomics programs is to produce, at a higher rate, protein samples. The Structural Biology and Genomics Department (IGBMC – Illkirch – France) is implicated in a structural genomics program of high eukaryotes whose goal is solving crystal structures of proteins and their complexes (including large complexes) related to human health and biotechnology. To achieve such a challenging goal, the Department has established a medium-throughput pipeline for producing protein samples suitable for structural biology studies. Here, we describe the setting up of our initiative from cloning to crystallization and we demonstrate that structural genomics may be manageable by academic laboratories by strategic investments in robotic and by adapting classical bench protocols and new developments, in particular in the field of protein expression, to parallelization.     

Transgenic production of epoxy fatty acids by expression of a cytochrome P450 enzyme from Euphorbia lagascae seed

Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Delta(12)-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Delta(12)-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developing E. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to the CYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Delta(9,15)) in cultures supplied with alpha-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Delta(9,15). Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Delta(12)-epoxy group of vernolic acid.