Understanding fatty acid synthesis in developing maize embryos using metabolic flux analysis

The efficiency with which developing maize embryos convert substrates into seed storage reserves was determined to be 57–71%, by incubating developing maize embryos with uniformly labeled ¹⁴C substrates and measuring their conversion to CO₂ and biomass products. To map the pattern of metabolic fluxes underlying this efficiency, maize embryos were labeled to isotopic steady state using a combination of labeled ¹³C-substrates. Intermediary metabolic fluxes were estimated by computer-aided modeling of the central metabolic network using the labeling data collected by NMR and GC-MS and the biomass composition. The resultant flux map reveals that even though 36% of the entering carbon goes through the oxidative pentose-phosphate pathway, this does not fully meet the NADPH demands for fatty acid synthesis. Metabolic flux analysis and enzyme activities highlight the importance of plastidic NADP-dependent malic enzyme, which provides one-third of the carbon and NADPH required for fatty acid synthesis in developing maize embryos.     

Regulation of the Nitrogen Transfer Pathway in the Arbuscular Mycorrhizal Symbiosis: Gene Characterization and the Coordination of Expression with Nitrogen Flux

The arbuscular mycorrhiza (AM) brings together the roots of over 80% of land plant species and fungi of the phylum Glomeromycota and greatly benefits plants through improved uptake of mineral nutrients. AM fungi can take up both nitrate and ammonium from the soil and transfer nitrogen (N) to host roots in nutritionally substantial quantities. The current model of N handling in the AM symbiosis includes the synthesis of arginine in the extraradical mycelium and the transfer of arginine to the intraradical mycelium, where it is broken down to release N for transfer to the host plant. To understand the mechanisms and regulation of N transfer from the fungus to the plant, 11 fungal genes putatively involved in the pathway were identified from Glomus intraradices, and for six of them the full-length coding sequence was functionally characterized by yeast complementation. Two glutamine synthetase isoforms were found to have different substrate affinities and expression patterns, suggesting different roles in N assimilation. The spatial and temporal expression of plant and fungal N metabolism genes were followed after nitrate was added to the extraradical mycelium under N-limited growth conditions using hairy root cultures. In parallel experiments with ¹⁵N, the levels and labeling of free amino acids were measured to follow transport and metabolism. The gene expression pattern and profiling of metabolites involved in the N pathway support the idea that the rapid uptake, translocation, and transfer of N by the fungus successively trigger metabolic gene expression responses in the extraradical mycelium, intraradical mycelium, and host plant.The arbuscular mycorrhizal (AM) symbiosis brings together the roots of the majority of land plant species and fungi of the phylum Glomeromycota to great mutual advantage (Smith and Read, 2008). AM fungi improve the acquisition of phosphate, nitrogen (N), sulfur, and trace elements such as copper and zinc (Clark and Zeto, 2000; Allen and Shachar-Hill, 2008) and increase the biotic and abiotic stress resistance of their host (Smith et al., 2010). In return, the host transfers up to 20% of its photosynthetically fixed carbon to the AM fungus (Jakobsen and Rosendahl, 1990), which depends on its host plant for its carbon supply (Bago et al., 2000).N is the nutrient whose availability most commonly limits plant growth in natural ecosystems. AM fungi can take up NO₃⁻NH₄⁺ and can also increase access to organic N sources from the soil (Ames et al., 1983; Johansen et al., 1993; Bago et al., 1996; Hodge et al., 2001). The translocation by the fungus can represent a significant route for N uptake by the plant (Johansen and Jensen, 1996). For example, Toussaint et al. (2004) showed that in an in vitro mycorrhiza at least 21% of the total N uptake in the roots came from the fungal extraradical mycelium (ERM); for other mycorrhizal systems, even larger proportions have been described (more than 30% and 50%; Govindarajulu et al., 2005; Jin et al., 2005). Tanaka and Yano (2005) reported that 75% of the N in leaves of mycorrhizal maize (Zea mays) was taken up by the ERM of Glomus aggregatum.A mechanism of N transfer from the fungus to the plant has been proposed (Bago et al., 2001) that involves the operation of a novel metabolic route in which N was translocated from the ERM to the intraradical mycelium (IRM) as Arg but transferred to the plant without carbon as inorganic N. This mechanism has been supported by labeling experiments (Johansen et al., 1996; Govindarajulu et al., 2005; Jin et al., 2005), enzyme activity analysis (Cruz et al., 2007), and limited gene expression data (Govindarajulu et al., 2005; Gomez et al., 2009; Guether et al., 2009). Nevertheless, our molecular knowledge of the metabolic and transport pathways involved and how they are regulated is still rudimentary. A better understanding of the mechanism and regulation of N uptake assimilation, translocation, and transfer to the host is important for potential applications of AM fungi as biofertilizers, bioprotectors, and bioregulators in sustainable agriculture and restoration as well as for understanding the role of AM fungi in natural ecosystems (Bruns et al., 2008).In this study, we postulate that the uptake, translocation, and transfer of N by the fungus triggers the metabolic gene expression responses successively in the ERM, IRM, and host plant, which will result in the synthesis and accumulation of Arg in the ERM, the turnover of Arg to release ammonium in the IRM, and the assimilation of ammonium by the host plant via the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway inside the root (Fig. 1). To test these predictions, 11 genes involved in the N primary assimilation and metabolism were cloned and verified from Glomus intraradices; six enzymes with full-length coding sequences (CDSs) were functionally characterized by yeast knockout mutant complementation. Two GS proteins were found to have different substrate affinities and expression patterns, suggesting that they have different roles in N assimilation. The time courses of gene expression and N movement in fungal and host tissues were analyzed following nitrate supply to the ERM of a mycorrhiza grown under N-limited conditions. The results substantially increase our knowledge of the identity and regulation of most of the metabolic and transport genes involved in N movement through the AM symbiosis.Open in a separate windowFigure 1.Working model of N transport and metabolism in the symbiosis between plant roots and arbuscular mycorrhizal fungi. N moves (black arrows) from the soil into the fungal ERM, through a series of metabolic conversion reactions into Arg, which is transported into the IRM within the root (host) and there is broken down; N is transferred to and assimilated by the host as ammonia. Red circles refer to the sites of action of the genes identified and analyzed in this study. Blue arrows indicate mechanisms hypothesized to regulate gene expression by N metabolites involved in the pathway.     

Host-finding behaviour and navigation capabilities of symbiotic zooxanthellae

Past studies have shown that the initiation of symbiosis between the Red-Sea soft coral Heteroxenia fuscescens and its symbiotic dinoflagellates occurs due to the chemical attraction of the motile algal cells to substances emanating from the coral polyps. However, the resulting swimming patterns of zooxanthellae have not been previously studied. This work examined algal swimming behaviour, host location and navigation capabilities under four conditions: (1) still water, (2) in still water with waterborne host attractants, (3) in flowing water, and (4) in flow with host attractants. Algae were capable of actively and effectively locating their host in still water as well as in flow. When in water containing host attractants, swimming became slower, motion patterns straighter and the direction of motion was mainly towards the host—even if this meant advancing upstream against flow velocities of up to 0.5 mm s⁻¹. Coral-algae encounter probability decreased the further downstream of the host algae were located, probably due to diffusion of the chemical signal. The results show how the chemoreceptive zooxanthellae modify their swimming pattern, direction, velocity, circuity and turning rate to accommodate efficient navigation in changing environmental conditions.     

Ten years of bacterial genome sequencing: comparative-genomics-based discoveries

It has been more than 10 years since the first bacterial genome sequence was published. Hundreds of bacterial genome sequences are now available for comparative genomics, and searching a given protein against more than a thousand genomes will soon be possible. The subject of this review will address a relatively straightforward question: “What have we learned from this vast amount of new genomic data?” Perhaps one of the most important lessons has been that genetic diversity, at the level of large-scale variation amongst even genomes of the same species, is far greater than was thought. The classical textbook view of evolution relying on the relatively slow accumulation of mutational events at the level of individual bases scattered throughout the genome has changed. One of the most obvious conclusions from examining the sequences from several hundred bacterial genomes is the enormous amount of diversity—even in different genomes from the same bacterial species. This diversity is generated by a variety of mechanisms, including mobile genetic elements and bacteriophages. An examination of the 20 Escherichia coli genomes sequenced so far dramatically illustrates this, with the genome size ranging from 4.6 to 5.5 Mbp; much of the variation appears to be of phage origin. This review also addresses mobile genetic elements, including pathogenicity islands and the structure of transposable elements. There are at least 20 different methods available to compare bacterial genomes. Metagenomics offers the chance to study genomic sequences found in ecosystems, including genomes of species that are difficult to culture. It has become clear that a genome sequence represents more than just a collection of gene sequences for an organism and that information concerning the environment and growth conditions for the organism are important for interpretation of the genomic data. The newly proposed Minimal Information about a Genome Sequence standard has been developed to obtain this information.     

A sequence-based filtering method for ncRNA identification and its application to searching for riboswitch elements

MOTIVATION: Recent studies have uncovered an "RNA world", in which non coding RNA (ncRNA) sequences play a central role in the regulation of gene expression. Computational studies on ncRNA have been directed toward developing detection methods for ncRNAs. State-of-the-art methods for the problem, like covariance models, suffer from high computational cost, underscoring the need for efficient filtering approaches that can identify promising sequence segments and speedup the detection process. RESULTS: In this paper we make several contributions toward this goal. First, we formalize the concept of a filter and provide figures of merit that allow comparison between filters. Second, we design efficient sequence based filters that dominate the current state-of-the-art HMM filters. Third, we provide a new formulation of the covariance model that allows speeding up RNA alignment. We demonstrate the power of our approach on both synthetic data and real bacterial genomes. We then apply our algorithm to the detection of novel riboswitch elements from the whole bacterial and archaeal genomes. Our results point to a number of novel riboswitch candidates, and include genomes that were not previously known to contain riboswitches. AVAILABILITY: The program is available upon request from the authors.     

GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum

Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94 shares many biochemical features with other HSP90 proteins, in particular its domain structure and ATPase activity, but also displays distinct activities, such as calcium binding, necessitated by the conditions in the endoplasmic reticulum. GRP94's mode of action varies from the general HSP90 theme in the conformational changes induced by nucleotide binding, and in its interactions with co-chaperones, which are very different from known cytosolic co-chaperones. GRP94 is more selective than many of the ER chaperones and the basis for this selectivity remains obscure. Recent development of molecular tools and functional assays has expanded the spectrum of clients that rely on GRP94 activity, but it is still not clear how the chaperone binds them, or what aspect of folding it impacts. These mechanistic questions and the regulation of GRP94 activity by other proteins and by post-translational modification differences pose new questions and present future research avenues. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Processing DNA molecules as text

Polymerase Chain Reaction (PCR) is the DNA-equivalent of Gutenberg’s movable type printing, both allowing large-scale replication of a piece of text. De novo DNA synthesis is the DNA-equivalent of mechanical typesetting, both ease the setting of text for replication. What is the DNA-equivalent of the word processor? Biology labs engage daily in DNA processing—the creation of variations and combinations of existing DNA—using a plethora of manual labor-intensive methods such as site-directed mutagenesis, error-prone PCR, assembly PCR, overlap extension PCR, cleavage and ligation, homologous recombination, and others. So far no universal method for DNA processing has been proposed and, consequently, no engineering discipline that could eliminate this manual labor has emerged. Here we present a novel operation on DNA molecules, called Y, which joins two DNA fragments into one, and show that it provides a foundation for DNA processing as it can implement all basic text processing operations on DNA molecules including insert, delete, replace, cut and paste and copy and paste. In addition, complicated DNA processing tasks such as the creation of libraries of DNA variants, chimeras and extensions can be accomplished with DNA processing plans consisting of multiple Y operations, which can be executed automatically under computer control. The resulting DNA processing system, which incorporates our earlier work on recursive DNA composition and error correction, is the first demonstration of a unified approach to DNA synthesis, editing, and library construction.

Electronic supplementary material

The online version of this article (doi:10.1007/s11693-010-9059-y) contains supplementary material, which is available to authorized users.     

Hyperbaric Oxygen Therapy Can Improve Post Concussion Syndrome Years after Mild Traumatic Brain Injury - Randomized Prospective Trial

Background

Traumatic brain injury (TBI) is the leading cause of death and disability in the US. Approximately 70-90% of the TBI cases are classified as mild, and up to 25% of them will not recover and suffer chronic neurocognitive impairments. The main pathology in these cases involves diffuse brain injuries, which are hard to detect by anatomical imaging yet noticeable in metabolic imaging. The current study tested the effectiveness of Hyperbaric Oxygen Therapy (HBOT) in improving brain function and quality of life in mTBI patients suffering chronic neurocognitive impairments.

Methods and Findings

The trial population included 56 mTBI patients 1–5 years after injury with prolonged post-concussion syndrome (PCS). The HBOT effect was evaluated by means of prospective, randomized, crossover controlled trial: the patients were randomly assigned to treated or crossover groups. Patients in the treated group were evaluated at baseline and following 40 HBOT sessions; patients in the crossover group were evaluated three times: at baseline, following a 2-month control period of no treatment, and following subsequent 2-months of 40 HBOT sessions. The HBOT protocol included 40 treatment sessions (5 days/week), 60 minutes each, with 100% oxygen at 1.5 ATA. “Mindstreams” was used for cognitive evaluations, quality of life (QOL) was evaluated by the EQ-5D, and changes in brain activity were assessed by SPECT imaging. Significant improvements were demonstrated in cognitive function and QOL in both groups following HBOT but no significant improvement was observed following the control period. SPECT imaging revealed elevated brain activity in good agreement with the cognitive improvements.

Conclusions

HBOT can induce neuroplasticity leading to repair of chronically impaired brain functions and improved quality of life in mTBI patients with prolonged PCS at late chronic stage.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00715052","term_id":"NCT00715052"}}NCT00715052