Sequence Editing Research Articles

Local search for the generalized tree alignment problem.

BackgroundA phylogeny postulates shared ancestry relationships among organisms in the form of a binary tree. Phylogenies attempt to answer an important question posed in biology: what are the ancestor-descendent relationships between organisms? At the core of every biological problem lies a phylogenetic component. The patterns that can be observed in nature are the product of complex interactions, constrained by the template that our ancestors provide. The problem of simultaneous tree and alignment estimation under Maximum Parsimony is known in combinatorial optimization as the Generalized Tree Alignment Problem (GTAP). The GTAP is the Steiner Tree Problem for the sequence edit distance. Like many biologically interesting problems, the GTAP is NP-Hard. Typically the Steiner Tree is presented under the Manhattan or the Hamming distances.ResultsExperimentally, the accuracy of the GTAP has been subjected to evaluation. Results show that phylogenies selected using the GTAP from unaligned sequences are competitive with the best methods and algorithms available. Here, we implement and explore experimentally existing and new local search heuristics for the GTAP using simulated and real data.ConclusionsThe methods presented here improve by more than three orders of magnitude in execution time the best local search heuristics existing to date when applied to real data.

The majority of endogenous microRNA targets within Alu elements avoid the microRNA machinery

The massive spread of repetitive elements in the human genome presents a substantial challenge to the organism, as such elements may accidentally contain seemingly functional motifs. A striking example is offered by the roughly one million copies of Alu repeats in the genome, of which ∼0.5% reside within genes' untranslated regions (UTRs), presenting ∼30 000 novel potential targets for highly conserved microRNAs (miRNAs). Here, we examine the functionality of miRNA targets within Alu elements in 3'UTRs in the human genome. Using a comprehensive dataset of miRNA overexpression assays, we show that mRNAs with miRNA targets within Alus are significantly less responsive to the miRNA effects compared with mRNAs that have the same targets outside Alus. Using Ago2-binding mRNA profiling, we confirm that the miRNA machinery avoids miRNA targets within Alus, as opposed to the highly efficient binding of targets outside Alus. We propose three features that prevent potential miRNA sites within Alus from being recognized by the miRNA machinery: (i) Alu repeats that contain miRNA targets and genuine functional miRNA targets appear to reside in distinct mutually exclusive territories within 3'UTRs; (ii) Alus have tight secondary structure that may limit access to the miRNA machinery; and (iii) A-to-I editing of Alu-derived mRNA sequences may divert miRNA targets. The combination of these features is proposed to allow toleration of Alu insertions into mRNAs. Nonetheless, a subset of miRNA targets within Alus appears not to possess any of the aforementioned features, and thus may represent cases where Alu insertion in the genome has introduced novel functional miRNA targets. Supplementary data are available at Bioinformatics online.

Triplex-forming Peptide Nucleic Acids Induce Heritable Elevations in Gamma-globin Expression in Hematopoietic Progenitor Cells

Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the β-globin subunit cause hemoglobinopathies such as sickle cell disease and β-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including β-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes.

Characterization of Peste des Petits Ruminants Virus, Eritrea, 2002–2011

Characterization of Peste des Petits Ruminants Virus, Eritrea, 2002–2011

A survey of enabling technologies in synthetic biology.

BackgroundRealizing constructive applications of synthetic biology requires continued development of enabling technologies as well as policies and practices to ensure these technologies remain accessible for research. Broadly defined, enabling technologies for synthetic biology include any reagent or method that, alone or in combination with associated technologies, provides the means to generate any new research tool or application. Because applications of synthetic biology likely will embody multiple patented inventions, it will be important to create structures for managing intellectual property rights that best promote continued innovation. Monitoring the enabling technologies of synthetic biology will facilitate the systematic investigation of property rights coupled to these technologies and help shape policies and practices that impact the use, regulation, patenting, and licensing of these technologies.ResultsWe conducted a survey among a self-identifying community of practitioners engaged in synthetic biology research to obtain their opinions and experiences with technologies that support the engineering of biological systems. Technologies widely used and considered enabling by survey participants included public and private registries of biological parts, standard methods for physical assembly of DNA constructs, genomic databases, software tools for search, alignment, analysis, and editing of DNA sequences, and commercial services for DNA synthesis and sequencing. Standards and methods supporting measurement, functional composition, and data exchange were less widely used though still considered enabling by a subset of survey participants.ConclusionsThe set of enabling technologies compiled from this survey provide insight into the many and varied technologies that support innovation in synthetic biology. Many of these technologies are widely accessible for use, either by virtue of being in the public domain or through legal tools such as non-exclusive licensing. Access to some patent protected technologies is less clear and use of these technologies may be subject to restrictions imposed by material transfer agreements or other contract terms. We expect the technologies considered enabling for synthetic biology to change as the field advances. By monitoring the enabling technologies of synthetic biology and addressing the policies and practices that impact their development and use, our hope is that the field will be better able to realize its full potential.

MiRGator v3.0: a microRNA portal for deep sequencing, expression profiling and mRNA targeting

Biogenesis and molecular function are two key subjects in the field of microRNA (miRNA) research. Deep sequencing has become the principal technique in cataloging of miRNA repertoire and generating expression profiles in an unbiased manner. Here, we describe the miRGator v3.0 update (http://mirgator.kobic.re.kr) that compiled the deep sequencing miRNA data available in public and implemented several novel tools to facilitate exploration of massive data. The miR-seq browser supports users to examine short read alignment with the secondary structure and read count information available in concurrent windows. Features such as sequence editing, sorting, ordering, import and export of user data would be of great utility for studying iso-miRs, miRNA editing and modifications. miRNA–target relation is essential for understanding miRNA function. Coexpression analysis of miRNA and target mRNAs, based on miRNA-seq and RNA-seq data from the same sample, is visualized in the heat-map and network views where users can investigate the inverse correlation of gene expression and target relations, compiled from various databases of predicted and validated targets. By keeping datasets and analytic tools up-to-date, miRGator should continue to serve as an integrated resource for biogenesis and functional investigation of miRNAs.

Functional remodeling of RNA processing in replacement chloroplasts by pathways retained from their predecessors.

Chloroplasts originate through the endosymbiotic integration of a host and a photosynthetic symbiont, with processes established within the host for the biogenesis and maintenance of the nascent chloroplast. It is thought that several photosynthetic eukaryotes have replaced their original chloroplasts with others derived from different source organisms in a process termed "serial endosymbiosis of chloroplasts." However, it is not known whether replacement chloroplasts are affected by the biogenesis and maintenance pathways established to support their predecessors. Here, we investigate whether pathways established during a previous chloroplast symbiosis function in the replacement chloroplasts of the dinoflagellate alga Karenia mikimotoi. We show that chloroplast transcripts in K. mikimotoi are subject to 3' polyuridylylation and extensive sequence editing. We confirm that these processes do not occur in free-living relatives of the replacement chloroplast lineage, but are otherwise found only in the ancestral, red algal-derived chloroplasts of dinoflagellates and their closest relatives. This indicates that these unusual RNA-processing pathways have been retained from the original symbiont lineage and made use of by the replacement chloroplast. Our results constitute an addition to current theories of chloroplast evolution in which chloroplast biogenesis may be radically remodeled by pathways remaining from previous symbioses.

Development of a custom 16S rRNA gene library for the identification and molecular subtyping of Salmonella enterica

The use of 16S rRNA gene sequencing within the regulatory workflow may help to reduce the time and labor involved in the identification and differentiation of Salmonella enterica isolates. However, a comprehensive, standardized reference library is needed in order to use this method with regulatory samples. The goal of this project was to acquire 16S rRNA partial and full gene sequences for a variety of S. enterica isolates and to use these sequences to build a custom 16S rRNA reference library. A total of 535 S. enterica isolates representing over 100 serotypes and 5 subspecies were selected for 16S rRNA partial gene sequencing (~500bp) and 66 isolates representing 32 serotypes and 2 subspecies were selected for 16S rRNA full gene sequencing (~1500bp). PCR, sequencing, and automated sequence assembly and editing were carried out using the MicroSEQ ID Microbial Identification System (Applied Biosystems). High quality sequences were obtained for 94.4% and 95.5% of the isolates sequenced over the partial and full genes, respectively. These sequences did not show sufficient divergence to reliably differentiate serotypes; however, they could be differentiated using 16S rRNA sequence typing based on intragenomic heterogeneity. A total of 83 unique 16S sequence types were obtained for use in the partial gene library and 58 unique 16S sequence types were obtained for entry into the full gene library. Preliminary sequencing results with one isolate analyzed in replicate were promising, with consistent matches to a specific 16S type in the custom library. The result of this study is a custom S. enterica 16S rRNA type library for potential use in the identification of isolates at the species, subspecies, and molecular subtype level. Further work will include validating the method for parameters such as exclusivity, sensitivity, and reproducibility.

RNA editing of protein sequences: A rare event in human transcriptomes

RNA editing, the post-transcriptional recoding of RNA molecules, has broad potential implications for gene expression. Several recent studies of human transcriptomes reported a high number of differences between DNA and RNA, including events not explained by any known mammalian RNA-editing mechanism. However, RNA-editing estimates differ by orders of magnitude, since technical limitations of high-throughput sequencing have been sometimes overlooked and sequencing errors have been confounded with editing sites. Here, we developed a series of computational approaches to analyze the extent of this process in the human transcriptome, identifying and addressing the major sources of error of a large-scale approach. We apply the detection pipeline to deep sequencing data from lymphoblastoid cell lines expressing ADAR1 at high levels, and show that noncanonical editing is unlikely to occur, with at least 85%-98% of candidate sites being the result of sequencing and mapping artifacts. By implementing a method to detect intronless gene duplications, we show that most noncanonical sites previously validated originate in read mismapping within these regions. Canonical A-to-G editing, on the other hand, is widespread in noncoding Alu sequences and rare in exonic and coding regions, where the validation rate also dropped. The genomic distribution of editing sites we find, together with the lack of consistency across studies or biological replicates, suggest a minor quantitative impact of this process in the overall recoding of protein sequences. We propose instead a primary role of ADAR1 protein as a defense system against elements potentially damaging to the genome.

BioMatriX: Sequence analysis, structure visualization, phylogenetics and linkage analysis workbench

The BioMatriX (Build Mine Xplore) is a bioinformatics work bench (http://www.bmx-biomatrix.blogspot.com) developed for biological science community to augment scientific research regarding genomics, proteomics, phylogenetics and linkage analysis in one platform. BioMatriX offers multi-functional services to perform specific tasks like DNA/RNA/Protein sequence analysis with graphical representations, sequence editing, sequence alignment, restriction enzyme mapping, protein structure visualization, mutation and structure superimposition programs along with phylogenetics tree construction supporting dendrograms, neighbor joining and unweighted pair group method with arithmetic mean (UPGMA) programs. Genomic studies like linkage programs are also implemented. Special emphasis has been paid to integrate all the resources in one software so that the researcher does not have to install numerous pieces of software to analyze his data. Keywords: Bioinformatics, linkage, visualizer, alignment, superimposition, phylogenetics

PyMod: sequence similarity searches, multiple sequence-structure alignments, and homology modeling within PyMOL

BackgroundIn recent years, an exponential growing number of tools for protein sequence analysis, editing and modeling tasks have been put at the disposal of the scientific community. Despite the vast majority of these tools have been released as open source software, their deep learning curves often discourages even the most experienced users.ResultsA simple and intuitive interface, PyMod, between the popular molecular graphics system PyMOL and several other tools (i.e., [PSI-]BLAST, ClustalW, MUSCLE, CEalign and MODELLER) has been developed, to show how the integration of the individual steps required for homology modeling and sequence/structure analysis within the PyMOL framework can hugely simplify these tasks. Sequence similarity searches, multiple sequence and structural alignments generation and editing, and even the possibility to merge sequence and structure alignments have been implemented in PyMod, with the aim of creating a simple, yet powerful tool for sequence and structure analysis and building of homology models.ConclusionsPyMod represents a new tool for the analysis and the manipulation of protein sequences and structures. The ease of use, integration with many sequence retrieving and alignment tools and PyMOL, one of the most used molecular visualization system, are the key features of this tool.Source code, installation instructions, video tutorials and a user's guide are freely available at the URL http://schubert.bio.uniroma1.it/pymod/index.html

Cloud BioLinux: pre-configured and on-demand bioinformatics computing for the genomics community

BackgroundA steep drop in the cost of next-generation sequencing during recent years has made the technology affordable to the majority of researchers, but downstream bioinformatic analysis still poses a resource bottleneck for smaller laboratories and institutes that do not have access to substantial computational resources. Sequencing instruments are typically bundled with only the minimal processing and storage capacity required for data capture during sequencing runs. Given the scale of sequence datasets, scientific value cannot be obtained from acquiring a sequencer unless it is accompanied by an equal investment in informatics infrastructure.ResultsCloud BioLinux is a publicly accessible Virtual Machine (VM) that enables scientists to quickly provision on-demand infrastructures for high-performance bioinformatics computing using cloud platforms. Users have instant access to a range of pre-configured command line and graphical software applications, including a full-featured desktop interface, documentation and over 135 bioinformatics packages for applications including sequence alignment, clustering, assembly, display, editing, and phylogeny. Each tool's functionality is fully described in the documentation directly accessible from the graphical interface of the VM. Besides the Amazon EC2 cloud, we have started instances of Cloud BioLinux on a private Eucalyptus cloud installed at the J. Craig Venter Institute, and demonstrated access to the bioinformatic tools interface through a remote connection to EC2 instances from a local desktop computer. Documentation for using Cloud BioLinux on EC2 is available from our project website, while a Eucalyptus cloud image and VirtualBox Appliance is also publicly available for download and use by researchers with access to private clouds.ConclusionsCloud BioLinux provides a platform for developing bioinformatics infrastructures on the cloud. An automated and configurable process builds Virtual Machines, allowing the development of highly customized versions from a shared code base. This shared community toolkit enables application specific analysis platforms on the cloud by minimizing the effort required to prepare and maintain them.

Automated sequence analysis and editing software for HIV drug resistance testing

BackgroundAccess to antiretroviral treatment in resource-limited-settings is inevitably paralleled by the emergence of HIV drug resistance. Monitoring treatment efficacy and HIV drugs resistance testing are therefore of increasing importance in resource-limited settings. Yet low-cost technologies and procedures suited to the particular context and constraints of such settings are still lacking. The ART-A (Affordable Resistance Testing for Africa) consortium brought together public and private partners to address this issue. ObjectivesTo develop an automated sequence analysis and editing software to support high throughput automated sequencing. Study designThe ART-A Software was designed to automatically process and edit ABI chromatograms or FASTA files from HIV-1 isolates. ResultsThe ART-A Software performs the basecalling, assigns quality values, aligns query sequences against a set reference, infers a consensus sequence, identifies the HIV type and subtype, translates the nucleotide sequence to amino acids and reports insertions/deletions, premature stop codons, ambiguities and mixed calls. The results can be automatically exported to Excel to identify mutations. Automated analysis was compared to manual analysis using a panel of 1624 PR–RT sequences generated in 3 different laboratories. Discrepancies between manual and automated sequence analysis were 0.69% at the nucleotide level and 0.57% at the amino acid level (668,047 AA analyzed), and discordances at major resistance mutations were recorded in 62 cases (4.83% of differences, 0.04% of all AA) for PR and 171 (6.18% of differences, 0.03% of all AA) cases for RT. ConclusionsThe ART-A Software is a time-sparing tool for pre-analyzing HIV and viral quasispecies sequences in high throughput laboratories and highlighting positions requiring attention.

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111. For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL<1000 copies/μl roughly increased in the last years (Fig. 3). The main steps for tropism testing (Fig. 4) demonstrated in this video: 1. Collection of a blood sample 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 3. Amplification of the env region 4. Amplification of the V3 region 5. Sequence reaction of the V3 amplicon 6. Purification of the sequencing samples 7. Sequencing the purified samples 8. Sequence editing 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 coreceptor usage (tropism) by sequence analysis using a genotypic approach.

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 2011. For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3). This region carries enough information to perform a reliable prediction. The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL < 1000 copies/μl roughly increased in the last years (Fig. 3). The main steps for tropism testing (Fig. 4) demonstrated in this video: Collection of a blood sample Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells Amplification of the env region Amplification of the V3 region Sequence reaction of the V3 amplicon Purification of the sequencing samples Sequencing the purified samples Sequence editing Sequencing data interpretation and tropism prediction.

Toward knowing the whole human: next-generation sequencing for personalized medicine.

The sequencing of the human genome, combined with brilliant technical advances in microarrays and computing, opened the genomic era of personalized medicine. The next generation of genomics is now being driven by massively parallel sequencers that are effectively high definition genetic analyzers capable of sequencing an entire human genome 30-times over in approximately a week for several thousand US dollars. Likewise, these next-generation sequencers, sometimes called deep sequencers, can sequence RNA transcriptomes to render unprecedented, high definition views of transcript sequence, SNP haplotypes, rare variants, splicing, exon boundaries and RNA editing. Presently, next-generation sequencing platforms can be grouped into 'discovery' platforms, which provide broad sequence coverage, but require days per sample, versus 'diagnostic' platforms, which provide a fraction of the coverage, but require only hours for sequencing. As these technologies converge, it will be possible to sequence a human genome in a matter of hours for a few hundred US dollars. While presenting considerable technical challenges in handling the massive data generated, next-generation sequencing platforms offer unparalleled opportunities for biological insights, target discovery and clinical diagnostics to accelerate personalized medicine in the coming years.

Graphical interface for motion editing using procedure visualization

In this paper, we introduce an interface for motion editing that visualizes editing procedures. For intuitive understanding and construction of motion editing procedures, our system represents an editing element that produces modified motion from a given input motion as a graphical node, and enables user to construct a whole motion editing procedure by connecting the nodes with a few mouse interactions. The system provides both the 3D transform manipulator and the time-line slider for intuitive controlling of the parameters of each node. The user evaluation results show that our system is easy and intuitive to be used by users for editing of motion sequences.

APOBEC3H haplotypes and HIV-1 pro-viral vif DNA sequence diversity in early untreated human immunodeficiency virus–1 infection

We examined single nucleotide polymorphisms (SNP) in the APOBEC3 locus on chromosome 22, paired with population sequences of pro-viral human immunodeficiency virus–1 (HIV-1) vif from peripheral blood mononuclear cells, from 96 recently HIV-1–infected treatment-naive adults. We found evidence for the existence of an APOBEC3H linkage disequilibrium (LD) block associated with variation in GA→AA, or APOBEC3F/H signature, sequence changes in pro-viral HIV-1 vif sequence (top 10 significant SNPs with a significant p = 4.8 × 10 −3). We identified a common five position risk haplotype distal to APOBEC3H (A3Hrh). These markers were in high LD (D′ = 1; r 2 = 0.98) to a previously described A3H “RED” haplotype containing a variant (E121) with enhanced susceptibility to HIV-1 Vif. This association was confirmed by a haplotype analysis. Homozygote carriers of the A3Hrh had lower GA->AA (A3F/H) sequence editing upon pro-viral HIV-1 vif sequence ( p = 0.01), and lower HIV-1 RNA levels over time during early, untreated HIV-1 infection, ( p = 0.015 mixed effects model). This effect may be due to enhanced susceptibility of A3H forms to HIV-1 Vif mediated viral suppression of sequence editing activity, slowing viral diversification and escape from immune responses.

Characterization and Expression Analysis of Four Glycine-Rich RNA-Binding Proteins Involved in Osmotic Response in Tobacco ( Nicotiana tabacum cv. Xanthi)

Plants have developed many signals and specific genes' regulations at both transcriptional and post-transcriptional levels in order to tolerate and adapt to various environmental stresses. RNA-binding proteins (RBPs) play crucial roles in the post-transcriptional regulation via mRNA splicing, polyadenylation, sequence editing, transport, mRNA stability, mRNA localization, and translation. In this paper, four cDNAs of glycine-rich RNA-binding proteins (GR-RBPs), named NtRGP-1a, -1b, -2, and -3, were isolated from Nicotiana tabacum by RT-PCR analysis, and special emphases were given to the sequences alignment, phylogenetic analysis and gene expression. Sequences alignment revealed minor difference of cDNA sequences, but no difference of deduced proteins between N. sylvestris and N. tabacum. Phylogenetic alignment revealed that four cDNAs in tobacco were clustered into two different groups. NtRGP-2 and -3 were evolutionarily closest to Arabidopsis GR-RBPs genes and related to animal GR-RBPs genes, while NtRGP-1a and -1b were closest to Gramineae GR-RBPs genes. The expression analyses of these four NtRGPs in response to different abiotic stressess showed the similar expression pattern. Moreover, the four NtRGPs, especially NtRGP-1a and NtRGP-3, were strongly induced by stresses including water, wound, cold, and high temperature, weakly induced by PEG, drought and SA, while reduced by NaCl and unaffected by ABA treatment. The fact that all of these abiotic stresses included in our experiments affected the water balance and resulted in osmotic stress on cellular level, suggests that NtRGPs in tobacco should be a family of crucial osmosis-related proteins, and may play a key role in signal transduction with ABA-independent pathway under abiotic stresses.

Edited MRS is sensitive to changes in lactate concentration during inspiratory hypoxia

To demonstrate the application of Mescher-Garwood (MEGA) point-resolved spectroscopy sequence (PRESS) editing to the detection of lactate in the brain at 3T and to investigate changes in lactate concentration associated with inspiratory gas challenges. Edited lactate measurements were made in six healthy volunteers while the subjects breathed normoxic (21% O(2)), hypoxic (12% O(2)), and hyperoxic (40% O(2)) gas mixtures. Lactate concentration was quantified relative to the unsuppressed water signal from the same volume. Lactate concentration was elevated in all subjects during hypoxia in a highly significant fashion (mean increase = 39%; P = 0.0003). There was no significant change seen in hyperoxia. MEGA-PRESS editing at 3T is sufficiently sensitive to detect lactate in the healthy brain with good signal-to-noise ratio (SNR), and can be used to investigate changes in cerebral metabolism arising during inspiratory gas challenges.