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PubMed2005

Lotus japonicus metabolic profiling. Development of gas chromatography-mass spectrometry resources for the study of plant-microbe interactions.

Symbiotic nitrogen fixation (SNF) in legume root nodules requires differentiation and integration of both plant and bacterial metabolism. Classical approaches of biochemistry, molecular biology, and genetics have revealed many aspects of primary metabolism in legume nodules that underpin SNF. Functional genomics approaches, especially transcriptomics and proteomics, are beginning to provide a more holistic picture of the metabolic potential of nodules in model legumes like Medicago truncatula and Lotus japonicus. To extend these approaches, we have established protocols for nonbiased measurement and analysis of hundreds of metabolites from L. japonicus, using gas chromatography coupled with mass spectrometry. Following creation of mass spectral tag libraries, which represent both known and unknown metabolites, we measured and compared relative metabolite levels in nodules, roots, leaves, and flowers of symbiotic plants. Principal component analysis of the data revealed distinct metabolic phenotypes for the different organs and led to the identification of marker metabolites for each. Metabolites that were enriched in nodules included: octadecanoic acid, asparagine, glutamate, homoserine, cysteine, putrescine, mannitol, threonic acid, gluconic acid, glyceric acid-3-P, and glycerol-3-P. Hierarchical cluster analysis enabled discrimination of 10 groups of metabolites, based on distribution patterns in diverse Lotus organs. The resources and tools described here, together with ongoing efforts in the areas of genome sequencing, and transcriptome and proteome analysis of L. japonicus and Mesorhizobium loti, should lead to a better understanding of nodule metabolism that underpins SNF.

Gas Chromatography-Mass Spectrometry↗

PubMed1999

The mammalian gene collection.

The Mammalian Gene Collection (MGC) project is a new effort by the NIH to generate full-length complementary DNA (cDNA) resources. This project will provide publicly accessible resources to the full research community. The MGC project entails the production of libraries, sequencing, and database and repository development, as well as the support of library construction, sequencing, and analytic technologies dedicated to the goal of obtaining a full set of human and other mammalian full-length (open reading frame) sequences and clones of expressed genes.

Animals↗

PubMed2006

Using ESTs to improve the accuracy of de novo gene prediction.

BACKGROUND: ESTs are a tremendous resource for determining the exon-intron structures of genes, but even extensive EST sequencing tends to leave many exons and genes untouched. Gene prediction systems based exclusively on EST alignments miss these exons and genes, leading to poor sensitivity. De novo gene prediction systems, which ignore ESTs in favor of genomic sequence, can predict such "untouched" exons, but they are less accurate when predicting exons to which ESTs align. TWINSCAN is the most accurate de novo gene finder available for nematodes and N-SCAN is the most accurate for mammals, as measured by exact CDS gene prediction and exact exon prediction. RESULTS: TWINSCAN_EST is a new system that successfully combines EST alignments with TWINSCAN. On the whole C. elegans genome TWINSCAN_EST shows 14% improvement in sensitivity and 13% in specificity in predicting exact gene structures compared to TWINSCAN without EST alignments. Not only are the structures revealed by EST alignments predicted correctly, but these also constrain the predictions without alignments, improving their accuracy. For the human genome, we used the same approach with N-SCAN, creating N-SCAN_EST. On the whole genome, N-SCAN_EST produced a 6% improvement in sensitivity and 1% in specificity of exact gene structure predictions compared to N-SCAN. CONCLUSION: TWINSCAN_EST and N-SCAN_EST are more accurate than TWINSCAN and N-SCAN, while retaining their ability to discover novel genes to which no ESTs align. Thus, we recommend using the EST versions of these programs to annotate any genome for which EST information is available.TWINSCAN_EST and N-SCAN_EST are part of the TWINSCAN open source software package http://genes.cse.wustl.edu/distribution/download_TS.html.

Algorithms↗

PubMed2026

Syndromic cholera diagnosis masks diverse causes of diarrhoeal disease in Burundi revealed by portable metagenomics.

BACKGROUND: Cholera outbreaks remain a major public-health challenge in sub-Saharan Africa, where diagnostic capacity is limited and clinical case definitions are non-specific and re ly heavily on syndromic diagnosis. Rapid identification of Vibrio cholerae is critical, yet cholera-suspected diarrhoea can have multiple infectious causes not captured by targeted diagnostics. METHODS: We evaluated a mobile, culture-independent metagenomic sequencing workflow for on-site detection of gastrointestinal pathogens directly from faecal samples in Burundi. The offline workflow combined long-read Oxford Nanopore Technologies (ONT) sequencing with rapid, laptop-based taxonomic and antimicrobial resistance (AMR) screening and was deployed across a health centre, a district hospital, and a refugee transit camp. The frontline and real-time results were verified using both conventional culturing and in-depth bioinformatic analyses. RESULTS: V. cholerae signals were only detected in a subset of suspected cholera cases, while many samples were dominated by alternative bacterial taxa, most frequently Escherichia coli. V. cholerae abundance correlated strongly with detection of the C holera T oxin P hage CTXφ, supporting differentiation between toxigenic signal and background exposure. AMR genes were detected across samples, providing early situational insight into resistance determinants among gastrointestinal bacteria. CONCLUSIONS: Mobile, offline metagenomic sequencing enables rapid frontline characterization of gastrointestinal disease, especially cholera-suspected, in resource-limited settings and complements existing diagnostics by improving etiological resolution and outbreak response.

Humans↗

PubMed2002

[Pediatric trauma life support and cardiopulmonary resuscitation].

Accidents are the most frequent cause of mortality among children older than one year. Thus, the need to proceed to cardiopulmonary resuscitation (CPR) during the early phases of trauma life support (TLS) is always a possibility. Trauma is a special situation in CPR: expected problems (i.e., hemorrhage, pneumo-hemothorax, hypothermia, and difficult intubation and vascular access), specific therapeutic actions (i.e., helmet retrieval and cervical spine immobilization), and exceptions to standard CPR guidelines (i.e., contraindication for the head tilt-chin lift manoeuvre) can arise. Therefore, TLS and CPR interventions must be appropriately integrated. TLS is considered a method (much like CPR). It combines organization and leadership with competent, structured and timely actions. Appropriate intervention within the first few moments ("platinum half-hour" and " golden hour") and first day ("silver day") is essential. As in CPR, two modalities can be distinguished: basic TLS (on the scene, without technical resources) and advanced TLS (with resources). The acronym PAA summarizes basic TLS: Protect-Alert-Aid. The advanced TLS sequence includes the following: primary survey and initial stabilization, secondary survey, triage, transport, and definitive care. The main objective of the primary survey and initial stabilization phase is the identification and treatment of injuries with immediate potential to cause death. CPR in the context of TLS should be adapted to the special features of trauma. Particular attention should be paid to the cervical spine. While not specific for trauma care, the early and generous administration of oxygen should be emphasized.

Cardiopulmonary Resuscitation↗

PubMed2006

Iterative ACORN as a high throughput tool in structural genomics.

High throughput macromolecular structure determination is very essential in structural genomics as the available number of sequence information far exceeds the number of available 3D structures. ACORN, a freely available resource in the CCP4 suite of programs is a comprehensive and efficient program for phasing in the determination of protein structures, when atomic resolution data are available. ACORN with the automatic model-building program ARP/wARP and refinement program REFMAC is a suitable combination for the high throughput structural genomics. ACORN can also be run with secondary structural elements like helices and sheets as inputs with high resolution data. In situations, where ACORN phasing is not sufficient for building the protein model, the fragments (incomplete model/dummy atoms) can again be used as a starting input. Iterative ACORN is proved to work efficiently in the subsequent model building stages in congerin (PDB-ID: lis3) and catalase (PDB-ID: 1gwe) for which models are available.

Animals↗

PubMed2006

Phylogeography of Brachyplatystoma rousseauxii (Siluriformes - Pimelodidae) in the Amazon Basin offers preliminary evidence for the first case of "homing" for an Amazonian migratory catfish.

The large pimelodid, Brachyplatystoma rousseauxii, is one of the two most important catfish species for the fisheries in the Amazon. It is captured by commercial and artisanal fishing fleets in at least five Amazonian countries, at fishing grounds more than 5000 km apart. Current evidence suggests a complex life cycle that includes the longest reproductive migration known for a freshwater fish species. Experimental fisheries have pointed to a decrease in yield in the Western Amazon. However, reliable information about the capture and status of this fishery resource is still nonexistent, and no study has ever addressed its genetic diversity. We sequenced the entire D-loop of 45 individuals of B. rousseauxii, fifteen from each of three different fishing locations along the main channel of the Solimões-Amazonas System covering a distance of around 2200 km. Results of phylogenetic analyses, molecular diversity estimations, analysis of molecular variance, and nested clade analysis, together show that there is no genetic segregation associated with location in the main channel, as one would expect for a migratory species. However, the significant decrease found in genetic diversity towards the western part of the Amazon could be explained by a non-random choice of tributary to spawn. It is possible that the genetic diversity of the migrating schools decreases towards the west because portions of the species' genetic diversity are being "captured" by the different effluents, as the fish migrates to spawn in the headwaters. Like the salmon in North America, B. rousseauxii may be returning to their home tributary to spawn.

Animal Migration↗

PubMed1997

The Human Genome Project: view from the Department of Energy.

The Human Genome Project (HGP), a research effort initiated by the Department of Energy (DOE) and jointly managed by the National Institutes of Health (NIH) and the DOE, will determine a representative (but complete) sequence of the DNA from a typical human cell. This information and the associated resources will help us understand the critical differences that make us individuals and lead to insights into many areas of medicine, biology, and biotechnology. The HGP was conceived because DOE and its predecessor agencies had a longstanding interest in developing more sensitive methods to detect genetic changes induced by ionizing radiation and to understand the related health effects. Additionally, the unique capabilities and resources of the DOE national laboratory system, including a multidisciplinary research environment, high-skill engineering and high-performance computing centers fostered the ideal conditions for the success of such an ambitious undertaking. Knowing the human genome will revolutionize the practice of biology in the next century with far-reaching implications to health care and sustainable development. In a novel departure from previous science programs, the Human Genome Project includes a subprogram devoted to the ethical, legal, and societal implications (ELSI) of human genome research.

Government Agencies↗

PubMed2005

Grains of knowledge: genomics of model cereals.

The economic and scientific importance of the cereals has motivated a rich history of research into their genetics, development, and evolution. The nearly completed sequence of the rice genome is emblematic of a transition to high-throughput genomics and computational biology that has also pervaded study of many other cereals. The relatively close (ca. <50 million years old) relationships among morphologically diverse cereals native to environments that sample much of global geographic diversity make the cereals particularly attractive for comparative studies of plant genome evolution. Extensive germplasm resources, largely a byproduct of their economic importance, together with growing collections of defined mutants, provide foundations for a host of post-genomic studies to shed more light on the relationship between sequence and function in this important group. Using the rapidly growing capabilities of several informatics resources, genomic data from model cereals are likely to be leveraged tremendously in the study and improvement of a wide range of crop plants that sustain much of the world's population, including many which still lack primary genomic resources.

DNA, Plant↗

PubMed2005

IMGT, the international ImMunoGeneTics information system.

The international ImMunoGeneTics information system (IMGT) (http://imgt.cines.fr), created in 1989, by the Laboratoire d'ImmunoGenetique Moleculaire LIGM (Universite Montpellier II and CNRS) at Montpellier, France, is a high-quality integrated knowledge resource specializing in the immunoglobulins (IGs), T cell receptors (TRs), major histocompatibility complex (MHC) of human and other vertebrates, and related proteins of the immune systems (RPI) that belong to the immunoglobulin superfamily (IgSF) and to the MHC superfamily (MhcSF). IMGT includes several sequence databases (IMGT/LIGM-DB, IMGT/PRIMER-DB, IMGT/PROTEIN-DB and IMGT/MHC-DB), one genome database (IMGT/GENE-DB) and one three-dimensional (3D) structure database (IMGT/3Dstructure-DB), Web resources comprising 8000 HTML pages (IMGT Marie-Paule page), and interactive tools. IMGT data are expertly annotated according to the rules of the IMGT Scientific chart, based on the IMGT-ONTOLOGY concepts. IMGT tools are particularly useful for the analysis of the IG and TR repertoires in normal physiological and pathological situations. IMGT is used in medical research (autoimmune diseases, infectious diseases, AIDS, leukemias, lymphomas, myelomas), veterinary research, biotechnology related to antibody engineering (phage displays, combinatorial libraries, chimeric, humanized and human antibodies), diagnostics (clonalities, detection and follow up of residual diseases) and therapeutical approaches (graft, immunotherapy and vaccinology). IMGT is freely available at http://imgt.cines.fr.

Animals↗

PubMed2000

A library-based bioinformatics services program.

Support for molecular biology researchers has been limited to traditional library resources and services in most academic health sciences libraries. The University of Washington Health Sciences Libraries have been providing specialized services to this user community since 1995. The library recruited a Ph.D. biologist to assess the molecular biological information needs of researchers and design strategies to enhance library resources and services. A survey of laboratory research groups identified areas of greatest need and led to the development of a three-pronged program: consultation, education, and resource development. Outcomes of this program include bioinformatics consultation services, library-based and graduate level courses, networking of sequence analysis tools, and a biological research Web site. Bioinformatics clients are drawn from diverse departments and include clinical researchers in need of tools that are not readily available outside of basic sciences laboratories. Evaluation and usage statistics indicate that researchers, regardless of departmental affiliation or position, require support to access molecular biology and genetics resources. Centralizing such services in the library is a natural synergy of interests and enhances the provision of traditional library resources. Successful implementation of a library-based bioinformatics program requires both subject-specific and library and information technology expertise.

Computational Biology↗

PubMed2025

Costs and cost-effectiveness of returning secondary findings from genomic sequencing based on the return of additional findings in the 100,000 Genomes Project.

PURPOSE: To assess costs and cost-effectiveness of returning additional findings from genome sequencing using data from the 100,000 Genomes Project (100kGP). METHODS: A model-based cost-utility analysis combining yield, consent rates, and cost data from the 100kGP with published estimates of downstream costs and quality-adjusted life years expected to accrue over a lifetime, after the identification of a pathogenic variant. RESULTS: The cost of returning additional findings to participants in the 100kGP was £7.1m or £81 per participant, with a yield of 0.85% for consented participants. The estimated lifetime incremental cost per participant was £125 and quality-adjusted life years 0.004, giving an incremental cost-effectiveness ratio of £28,830. Implementing a policy of returning additional findings is unlikely to be cost-effective (ie, 13%) at a willingness-to-pay threshold of £20,000. A short-term cost of returning findings of £43 per participant or lower (compared with the base case of £81) would result in an incremental cost-effectiveness ratio of less than £20,000. Alternatively, cost-effectiveness may be improved by returning additional findings to younger patient populations. CONCLUSION: Return of additional findings following genome sequencing for this group of conditions may not be a cost-effective use of health care system resources. Our cost-effectiveness outcomes rely on published estimates and should be validated through long-term follow-up data.

Humans↗

PubMed2005

Survey of current protein family databases and their application in comparative, structural and functional genomics.

The last two decades have witnessed significant expansions in the databases storing information on the sequences and structures of proteins. This has led to the creation of many excellent protein family resources, which classify proteins according to their evolutionary relationship. These have allowed extensive insights into evolution and particularly how protein function mutates and evolves over time. Such analyses have greatly assisted the inheritance of functional annotations between experimentally characterised and uncharacterised genes. Moreover, the development of bioinformatics tools acts as a companion to the new technologies emerging in biology, such as transcriptomics and proteomics. The latter enable researchers to analyse gene expression profiles and interactions on a genome-wide scale, generating vast datasets of proteins, many of which include experimentally uncharacterised proteins. Protein family/function databases can be used to help interpret this data and allow us to benefit more fully from these technologies. This review aims to summarise the most popular sequence- and structure-based protein family databases. We also cover their application to comparative genomics and the functional annotation of the genomes.

Biological Evolution↗

PubMed2006

Evaluation of simplified DNA extraction methods for emm typing of group A streptococci.

Simplified methods of DNA extraction for amplification and sequencing for emm typing of group A streptococci (GAS) can save valuable time and cost in resource crunch situations. To evaluate this, we compared two methods of DNA extraction directly from colonies with the standard CDC cell lysate method for emm typing of 50 GAS strains isolated from children with pharyngitis and impetigo. For this, GAS colonies were transferred into two sets of PCR tubes. One set was preheated at 94 degrees C for two minutes in the thermal cycler and cooled while the other set was frozen overnight at -20 degrees C and then thawed before adding the PCR mix. For the cell lysate method, cells were treated with mutanolysin and hyaluronidase before heating at 100 degrees C for 10 minutes and cooling immediately as recommended in the CDC method. All 50 strains could be typed by sequencing the hyper variable region of the emm gene after amplification. The quality of sequences and the emm types identified were also identical. Our study shows that the two simplified DNA extraction methods directly from colonies can conveniently be used for typing a large number of GAS strains easily in relatively short time.

Antigens, Bacterial↗

PubMed1998

Optimization of capillary chromatography ion trap-mass spectrometry for identification of gel-separated proteins.

The current paradigm for protein identification using mass spectrometric derived peptide-mass and fragment-ion data employs computer algorithms which match uninterpreted or partially interpreted fragment-ion data to sequence databases, both protein and translated nucleotide sequence databases. Nucleotide sequence databases continue to grow at a rapid rate for some species, providing an unsurpassed resource for protein identification in those species. Ion-trap mass spectrometers with their ability to rapidly generate fragment-ion spectra in a data-dependent manner with high sensitivity and accuracy has led to their increased use for protein identification. We have investigated various parameters on a commercial ion trap-mass spectrometer to enhance our ability to identify peptides separated by capillary reversed phase-high performance liquid chromatography (RP-HPLC) coupled on-line to the mass spectrometer. By systematically evaluating the standard parameters (ion injection time and number of microscans) together with selection of multiple ions from the full mass range, improved tandem mass spectrometry (MS/MS) spectra were generated, facilitating identification of proteins at a low pmol level. Application of this technology to the identification of a standard protein and an unknown from an affinity-enriched mixture are shown.

Amino Acid Sequence↗

PubMed1992

Interdependence of translation, transcription and mRNA degradation in the lacZ gene.

We have constructed a collection of Escherichia coli strains which differ by point mutations in the ribosome binding site (RBS) that drives the translation of the lacZ gene. These mutations affect the Shine-Dalgarno sequence or the initiation codon, or create secondary structures that sequester these elements, and result in a 200-fold variation in beta-galactosidase expression. Surprisingly, these variations of expression are paralleled by nearly equivalent changes in the lacZ mRNA level. The ratio of the beta-galactosidase expression to the mRNA level reflects the average spacing between translating ribosomes: hence, paradoxically, mutations that affect translation initiation do not correspondingly change this spacing. Further analysis of the mRNA level variations shows that they originate from two independent mechanisms. When beta-galactosidase expression exceeds a threshold corresponding roughly to one translation event per transcript, the variations in the efficiency of translation initiation affect largely the chemical and functional lifetimes of the mRNA. We further show that the rate-limiting step in the chemical decay process is an RNase E-dependent cleavage, which is outcompeted by translation initiation. Below this expression threshold, the mRNA lifetime levels out and strain-to-strain variations in mRNA level arise solely from polarity effects. We suggest that, in this activity range, most mRNA molecules that escape polarity are crossed by a single ribosome, and hence are identical from the viewpoint of degradation. Altogether, the tight couplings between translation initiation on one hand, polarity and/or mRNA degradation on the other, result in translation initiation events being closely spaced in time even from inefficient RBS, at the expense of the mRNA level. Finally, we evocate the possible beneficial consequences of a coupling between translation, transcription and mRNA degradation, for the management of cellular resources.

Base Sequence↗

PubMed2004

Types and frequencies of sequencing errors in methyl-filtered and high c0t maize genome survey sequences.

The Maize Genome Sequencing Consortium has deposited into GenBank more than 850,000 maize (Zea mays) genome survey sequences (GSSs) generated via two gene enrichment strategies, methylation filtration and high-C(0)t (HC) fractionation. These GSSs are a valuable resource for generating genome assemblies and the discovery of single nucleotide polymorphisms and nearly identical paralogs. Based on the rate of mismatches between 183 GSSs (105 methylation filtration + 78 HC) and 10 control genes, the rate of sequencing errors in these GSSs is 2.3 x 10(-3). As expected many of these errors were derived from insufficient vector trimming and base-calling errors. Surprisingly, however, some errors were due to cloning artifacts. These G.C to A.T transitions are restricted to HC clones; over 40% of HC clones contain at least one such artifact. Because it is not possible to distinguish the cloning artifacts from biologically relevant polymorphisms, HC sequences should be used with caution for the discovery of single nucleotide polymorphisms or paramorphisms. The average rate of sequencing errors was reduced 6-fold (to 3.6 x 10(-4)) by applying more stringent trimming parameters. This trimming resulted in the loss of only 11% of the bases (15,469/144,968). Due to redundancy among GSSs this more stringent trimming reduced coverage of promoters, exons, and introns by only 0%, 1%, and 4%, respectively. Hence, at the cost of a very modest loss of gene coverage, the quality of these maize GSSs can approach Bermuda standards, even prior to assembly.

DNA, Plant↗

PubMed1991

Extraction, purification, and characterization of major outer membrane proteins from Wolinella recta ATCC 33238.

The outer membrane of Wolinella recta ATCC 33238 was isolated by French pressure cell disruption and differential centrifugation. Outer membrane proteins (OMPs) were solubilized by Zwittergent 3.14 extraction and separated by DEAE-Sephacel ion-exchange chromatography. The major OMPs that were found in W. recta ATCC 33238 and in several other Wolinella spp. consisted of proteins with apparent molecular masses of 51, 45, and 43 kDa. These three conserved proteins were purified to essential homogeneity by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized chemically. Heating at between 75 and 100 degrees C revealed both the 43- and 51-kDa proteins to be heat modified from apparent molecular masses of 32 and 38 kDa, respectively. The 45-kDa protein was unmodified at all temperatures tested. Two-dimensional isoelectric focusing-SDS-PAGE revealed the 51-kDa protein to be composed of multiple pIs between a pH of 5.0 and greater than 8.0 while the 43- and 45-kDa proteins had a pI of approximately 6.0. N'-terminal amino acid sequence analysis of the first 30 to 40 amino acids and search of the Protein Identification Resource data base for similar proteins only revealed the 43-kDa protein to be similar to the P.69 OMP of Bordetella pertussis; however, the homology was weak (33%). Amino acid analysis revealed the 43-kDa protein to be noncharged and the 45- and 51-kDa proteins to be hydrophilic, containing between 38 to 42% polar residues but no cysteine. This study reports the purification and partial characterization of three conserved proteins in W. recta ATCC 33238.

Amino Acid Sequence↗