Publications by authors named "David M Kristensen"

54 Publications

Acetaminophen metabolism revisited using non-targeted analyses: Implications for human biomonitoring.

Environ Int 2021 Apr 29;149:106388. Epub 2021 Jan 29.

Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) - UMR_S 1085, F-35000 Rennes, France; Danish Headache Center, Department of Neurology, Rigshospitalet-Glostrup, Denmark.

The analgesic paracetamol/acetaminophen (N-acetyl-4-aminophenol, APAP) is commonly used to relieve pain, fever and malaise. While sales have increased worldwide, a growing body of experimental and epidemiological evidence has suggested APAP as a possible risk factor for various health disorders in humans. To perform internal exposure-based risk assessment, the use of accurate and optimized biomonitoring methods is critical. However, retrospectively assessing pharmaceutical use of APAP in humans is challenging because of its short half-life. The objective of this study was to address the key issue of potential underestimation of APAP use using current standard analytical methods based on urinary analyses of free APAP and its phase II conjugates. The question we address is whether investigating additional metabolites than direct phase II conjugates could improve the monitoring of APAP. Using non-targeted analyses based on high-resolution mass spectrometry, we identified, in a controlled longitudinal exposure study with male volunteers, overlooked APAP metabolites with delayed formation and excretion rates. We postulate that these metabolites are formed via the thiomethyl shunt after the enterohepatic circulation as already observed in rodents. Importantly, these conjugated thiomethyl metabolites were (i) of comparable diagnostic sensitivity as the free APAP and its phase II conjugates detected by current methods; (ii) had delayed peak levels in blood and urine compared to other APAP metabolites and therefore potentially extend the window of exposure assessment; and (iii) provide relevant information regarding metabolic pathways of interest from a toxicological point of view. Including these metabolites in future APAP biomonitoring methods therefore provides an option to decrease potential underestimation of APAP use. Moreover, our data challenge the notion that the standard methods in biomonitoring based exclusively on the parent compound and its phase II metabolites are adequate for human biomonitoring of a non-persistent chemical such as APAP.
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http://dx.doi.org/10.1016/j.envint.2021.106388DOI Listing
April 2021

NCBI's Virus Discovery Codeathon: Building "FIVE" -The Federated Index of Viral Experiments API Index.

Viruses 2020 12 10;12(12). Epub 2020 Dec 10.

National Center for Biotechnology Information, U.S. National Library of Medicine, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20894, USA.

Viruses represent important test cases for data federation due to their genome size and the rapid increase in sequence data in publicly available databases. However, some consequences of previously decentralized (unfederated) data are lack of consensus or comparisons between feature annotations. Unifying or displaying alternative annotations should be a priority both for communities with robust entry representation and for nascent communities with burgeoning data sources. To this end, during this three-day continuation of the Virus Hunting Toolkit codeathon series (VHT-2), a new integrated and federated viral index was elaborated. This Federated Index of Viral Experiments (FIVE) integrates pre-existing and novel functional and taxonomy annotations and virus-host pairings. Variability in the context of viral genomic diversity is often overlooked in virus databases. As a proof-of-concept, FIVE was the first attempt to include viral genome variation for HIV, the most well-studied human pathogen, through viral genome diversity graphs. As per the publication of this manuscript, FIVE is the first implementation of a virus-specific federated index of such scope. FIVE is coded in BigQuery for optimal access of large quantities of data and is publicly accessible. Many projects of database or index federation fail to provide easier alternatives to access or query information. To this end, a Python API query system was developed to enhance the accessibility of FIVE.
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http://dx.doi.org/10.3390/v12121424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764237PMC
December 2020

No additive effect of combining sumatriptan and olcegepant in the GTN mouse model of migraine.

Cephalalgia 2020 Oct 15:333102420963857. Epub 2020 Oct 15.

Department of Neurology, Danish Headache Center, Rigshospitalet Glostrup, University of Copenhagen, Glostrup, Denmark.

Introduction: Despite recent advances in migraine treatment there is a need for therapies with higher clinical efficacy and/or fewer side effects. Triptans (5-HT agonists) are essential in the present treatment regime and gepants (CGRP-receptor antagonists) are recognized as effective in acute migraine treatment. Triptans and gepants have different mechanisms of action and here we tested the hypothesis that a combination of these drugs (sumatriptan and olcegepant) would result in an additive effect.

Methods: Using the validated glyceryl trinitrate mouse model of migraine, we initially tested dose-response relationships of sumatriptan (0.1, 0.3, and 0.6 mg/kg IP) and olcegepant (0.25, 0.50, and 1.0 mg/kg IP) to find suitable high and low doses. Subsequently, we performed a combination study of the two drugs with a low and a high dose. All experiments were vehicle (placebo) controlled and blinded.

Results: Sumatriptan significantly reduced glyceryl trinitrate-induced allodynia (F(4,54) = 13.51,  < 0.0001) at all doses. Olcegepant also reduced glyceryl trinitrate-induced allodynia (F(4,53) = 16.11,  < 0.0001) with the two higher doses being significantly effective. Combining 0.50 mg/kg olcegepant with 0.1 or 0.6 mg/kg sumatriptan did not have any improved effect compared to either drug alone ( > 0.50 on all days) in our mouse model.

Conclusion: Combining olcegepant and sumatriptan did not have an additive effect compared to single-drug treatment in this study. Triptan-gepant combinations will therefore most likely not improve migraine treatment. Nevertheless, further studies are necessary, and combinations should also be examined in patients with migraine.
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http://dx.doi.org/10.1177/0333102420963857DOI Listing
October 2020

Functional gene networks reveal distinct mechanisms segregating in migraine families.

Brain 2020 10;143(10):2945-2956

Danish Headache Center, Department of Neurology, Rigshospitalet Glostrup, 2600 Glostrup, Denmark.

Migraine is the most common neurological disorder worldwide and it has been shown to have complex polygenic origins with a heritability of estimated 40-70%. Both common and rare genetic variants are believed to underlie the pathophysiology of the prevalent types of migraine, migraine with typical aura and migraine without aura. However, only common variants have been identified so far. Here we identify for the first time a gene module with rare mutations through a systems genetics approach integrating RNA sequencing data from brain and vascular tissues likely to be involved in migraine pathology in combination with whole genome sequencing of 117 migraine families. We found a gene module in the visual cortex, based on single nuclei RNA sequencing data, that had increased rare mutations in the migraine families and replicated this in a second independent cohort of 1930 patients. This module was mainly expressed by interneurons, pyramidal CA1, and pyramidal SS cells, and pathway analysis showed association with hormonal signalling (thyrotropin-releasing hormone receptor and oxytocin receptor signalling pathways), Alzheimer's disease pathway, serotonin receptor pathway and general heterotrimeric G-protein signalling pathways. Our results demonstrate that rare functional gene variants are strongly implicated in the pathophysiology of migraine. Furthermore, we anticipate that the results can be used to explain the critical mechanisms behind migraine and potentially improving the treatment regime for migraine patients.
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http://dx.doi.org/10.1093/brain/awaa242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780491PMC
October 2020

ATP sensitive potassium (K) channel inhibition: A promising new drug target for migraine.

Cephalalgia 2020 06 16;40(7):650-664. Epub 2020 May 16.

Danish Headache Center, Department of Neurology, Glostrup Research Institute, Righospitalet Glostrup, Glostrup, Denmark.

Background: Recently, the adenosine triphosphate (ATP) sensitive potassium channel opener levcromakalim was shown to induce migraine attacks with a far higher incidence than any previous provoking agent such as calcitonin gene-related peptide. Here, we show efficacy of ATP sensitive potassium channel inhibitors in two validated rodent models of migraine.

Methods: In female spontaneous trigeminal allodynic rats, the sensitivity of the frontal region of the head was tested by an electronic von Frey filament device. In mice, cutaneous hypersensitivity was induced by repeated glyceryl trinitrate or levcromakalim injections over nine days, as measured with von Frey filaments in the hindpaw. Release of calcitonin gene-related peptide from dura mater and trigeminal ganglion was studied .

Results: The ATP sensitive potassium channel inhibitor glibenclamide attenuated the spontaneous cephalic hypersensitivity in spontaneous trigeminal allodynic rats and glyceryl trinitrate-induced hypersensitivity of the hindpaw in mice. It also inhibited CGRP release from dura mater and the trigeminal ganglion isolated from spontaneous trigeminal allodynic rats. The hypersensitivity was also diminished by the structurally different ATP sensitive potassium channel inhibitor gliquidone. Mice injected with the ATP sensitive potassium channel opener levcromakalim developed a progressive hypersensitivity that was completely blocked by glibenclamide, confirming target engagement.

Conclusion: The results suggest that ATP sensitive potassium channel inhibitors could be novel and highly effective drugs in the treatment of migraine.
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http://dx.doi.org/10.1177/0333102420925513DOI Listing
June 2020

No central action of CGRP antagonising drugs in the GTN mouse model of migraine.

Cephalalgia 2020 08 29;40(9):924-934. Epub 2020 Mar 29.

Danish Headache Center, Glostrup Research Institute, Rigshospitalet Glostrup, Glostrup, Denmark.

Introduction: Clinically, calcitonin gene-related peptide antagonising drugs are recognized as effective in migraine treatment, but their site of action is debated. Only a small fraction of these compounds pass the blood-brain barrier and accesses the central nervous system. Regardless, it has been argued that the central nervous system is the site of action. Here, we test this hypothesis by bypassing the blood-brain barrier through intracerebroventricular injection of calcitonin gene-related peptide antagonising drugs.

Methods: We used the glyceryl trinitrate (GTN) mouse model, which is well validated by its response to specific migraine drugs. The calcitonin gene-related peptide receptor antagonist olcegepant and the calcitonin gene-related peptide monoclonal antibody ALD405 were administered either intraperitoneally or intracerebroventricularly. The outcome measure was cutaneous mechanical allodynia.

Results: Mice given olcegepant intraperitoneally + GTN on day 1 had a mean 50% withdrawal threshold of 1.2 g in contrast to mice receiving placebo + GTN, which had a threshold of 0.3 g ( < 0.001). Similarly, in the ALD405 + GTN group, mice had thresholds of 1.2 g versus 0.2 g in the placebo + GTN group ( < 0.001). However, both drugs were ineffective when delivered intracerebroventricularly, as control and active groups had identical mechanical sensitivity thresholds, 0.2 g versus 0.1 g and 0.1 g versus 0.1 g for olcegepant and ALD405, respectively ( > 0.99 in both cases).

Discussion: The site of action of olcegepant and of the monoclonal antibody ALD405 is outside the blood-brain barrier in this mouse model of migraine. It is likely that these results can be generalised to all gepants and all antibodies and that the results are relevant for human migraine.
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http://dx.doi.org/10.1177/0333102420914913DOI Listing
August 2020

Author Correction: Characterisation and localisation of the endocannabinoid system components in the adult human testis.

Sci Rep 2020 Jan 22;10(1):1267. Epub 2020 Jan 22.

Department of Growth & Reproduction, and EDMaRC, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-58153-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974603PMC
January 2020

Von Frey testing revisited: Provision of an online algorithm for improved accuracy of 50% thresholds.

Eur J Pain 2020 04 23;24(4):783-790. Epub 2020 Jan 23.

Danish Headache Center, Glostrup Research Institute, Rigshospitalet Glostrup, Denmark.

Background: In the pain field, it is essential to quantify nociceptive responses. The response to the application of von Frey filaments to the skin measures tactile sensitivity and is a surrogate marker of allodynia in states of peripheral and/or central sensitization. The method is widely used across species within the pain field. However, uncertainties appear to exist regarding the appropriate method for analysing obtained data. Therefore, there is a need for refinement of the calculations for transformation of raw data to quantifiable data.

Methods: Here, we briefly review the fundamentals behind von Frey testing using the standard up-down method and the associated statistics and show how different parameters of the statistical equation influence the calculated 50% threshold results. We discuss how to obtain the most accurate estimations in a given experimental setting.

Results: To enhance accuracy and reproducibility across laboratories, we present an easy to use algorithm that calculates 50% thresholds based on the exact filaments and their interval using math beyond the traditional methods. This tool is available to the everyday user of von Frey filaments and allows the insertion of all imaginable ranges of filaments and is thus applicable to data derived in any species.

Conclusion: We advocate for the use of this algorithm to minimize inaccuracies and to improve internal and external reproducibility.

Significance: The von Frey testing procedure is standard for assessing peripheral and central sensitization but is associated with inaccuracies and lack of transparency in the associated math. Here, we describe these problems and present a novel statistical algorithm that calculates the exact thresholds using math beyond the traditional methods. The online platform is transparent, free of charge and easy to use also for the everyday user of von Frey filaments. Application of this resource will ultimately reduce errors due to methodological misinterpretations and increase reproducibility across laboratories.
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http://dx.doi.org/10.1002/ejp.1528DOI Listing
April 2020

Characterisation and localisation of the endocannabinoid system components in the adult human testis.

Sci Rep 2019 09 19;9(1):12866. Epub 2019 Sep 19.

Department of Growth & Reproduction, and EDMaRC, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

Heavy use of cannabis (marijuana) has been associated with decreased semen quality, which may reflect disruption of the endocannabinoid system (ECS) in the male reproductive tract by exogenous cannabinoids. Components of ECS have been previously described in human spermatozoa and in the rodent testis but there is little information on the ECS expression within the human testis. In this study we characterised the main components of the ECS by immunohistochemistry (IHC) on archived testis tissue samples from 15 patients, and by in silico analysis of existing transcriptome datasets from testicular cell populations. The presence of 2-arachidonoylglycerol (2-AG) in the human testis was confirmed by matrix-assisted laser desorption ionization imaging analysis. Endocannabinoid-synthesising enzymes; diacylglycerol lipase (DAGL) and N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), were detected in germ cells and somatic cells, respectively. The cannabinoid receptors, CNR1 and CNR2 were detected at a low level in post-meiotic germ cells and Leydig- and peritubular cells. Different transcripts encoding distinct receptor isoforms (CB1, CB1A, CB1B and CB2A) were also differentially distributed, mainly in germ cells. The cannabinoid-metabolising enzymes were abundantly present; the α/β-hydrolase domain-containing protein 2 (ABHD2) in all germ cell types, except early spermatocytes, the monoacylglycerol lipase (MGLL) in Sertoli cells, and the fatty acid amide hydrolase (FAAH) in late spermatocytes and post-meiotic germ cells. Our findings are consistent with a direct involvement of the ECS in regulation of human testicular physiology, including spermatogenesis and Leydig cell function. The study provides new evidence supporting observations that recreational cannabis can have possible deleterious effects on human testicular function.
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http://dx.doi.org/10.1038/s41598-019-49177-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753062PMC
September 2019

Clarifying the link between ibuprofen and hypogonadism.

Nat Rev Urol 2019 03;16(3):201

Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) - UMR_S1085, F-35000 Rennes, France.

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http://dx.doi.org/10.1038/s41585-019-0146-xDOI Listing
March 2019

Microbial genome analysis: the COG approach.

Brief Bioinform 2019 07;20(4):1063-1070

For the past 20 years, the Clusters of Orthologous Genes (COG) database had been a popular tool for microbial genome annotation and comparative genomics. Initially created for the purpose of evolutionary classification of protein families, the COG have been used, apart from straightforward functional annotation of sequenced genomes, for such tasks as (i) unification of genome annotation in groups of related organisms; (ii) identification of missing and/or undetected genes in complete microbial genomes; (iii) analysis of genomic neighborhoods, in many cases allowing prediction of novel functional systems; (iv) analysis of metabolic pathways and prediction of alternative forms of enzymes; (v) comparison of organisms by COG functional categories; and (vi) prioritization of targets for structural and functional characterization. Here we review the principles of the COG approach and discuss its key advantages and drawbacks in microbial genome analysis.
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http://dx.doi.org/10.1093/bib/bbx117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781585PMC
July 2019

Reconstruction of the evolution of microbial defense systems.

BMC Evol Biol 2017 04 4;17(1):94. Epub 2017 Apr 4.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, 20894, USA.

Background: Evolution of bacterial and archaeal genomes is a highly dynamic process that involves intensive loss of genes as well as gene gain via horizontal transfer, with a lesser contribution from gene duplication. The rates of these processes can be estimated by comparing genomes that are linked by an evolutionary tree. These estimated rates of genome dynamics events substantially differ for different functional classes of genes. The genes involved in defense against viruses and other invading DNA are among those that are gained and lost at the highest rates.

Results: We employed a stochastic birth-and-death model to obtain maximum likelihood estimates of the rates of gain and loss of defense genes in 35 groups of closely related bacterial genomes and one group of archaeal genomes. We find that on average, the defense genes experience 1.4 fold higher flux than the rest of microbial genes. This excessive flux of defense genes over the genomic mean is consistent across diverse microbial groups. The few exceptions include intracellular parasites with small, degraded genomes that possess few defense systems which are more stable than in other microbes. Generally, defense genes follow the previously established pattern of genome dynamics, with gene family loss being about 3 times more common than gain and an order of magnitude more common than expansion or contraction of gene families. Case by case analysis of the evolutionary dynamics of defense genes indicates frequent multiple events in the same locus and widespread involvement of mobile elements in the gain and loss of defense genes.

Conclusions: Evolution of microbial defense systems is highly dynamic but, notwithstanding the host-parasite arms race, generally follows the same trends that have been established for the rest of the genes. Apart from the paucity and the low flux of defense genes in parasitic bacteria with deteriorating genomes, there is no clear connection between the evolutionary regime of defense systems and microbial life style.
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http://dx.doi.org/10.1186/s12862-017-0942-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379612PMC
April 2017

Ibuprofen results in alterations of human fetal testis development.

Sci Rep 2017 03 10;7:44184. Epub 2017 Mar 10.

Institut national de la santé et de la recherche médicale (Inserm), Institut de recherche en santé, environnement et travail (Irset - Inserm UMR 1085), Université de Rennes 1, 9 Avenue Léon Bernard, F-35000 RENNES, France.

Among pregnant women ibuprofen is one of the most frequently used pharmaceutical compounds with up to 28% reporting use. Regardless of this, it remains unknown whether ibuprofen could act as an endocrine disruptor as reported for fellow analgesics paracetamol and aspirin. To investigate this, we exposed human fetal testes (7-17 gestational weeks (GW)) to ibuprofen using ex vivo culture and xenograft systems. Ibuprofen suppressed testosterone and Leydig cell hormone INSL3 during culture of 8-9 GW fetal testes with concomitant reduction in expression of the steroidogenic enzymes CYP11A1, CYP17A1 and HSD17B3, and of INSL3. Testosterone was not suppressed in testes from fetuses younger than 8 GW, older than 10-12 GW, or in second trimester xenografted testes (14-17 GW). Ex vivo, ibuprofen also affected Sertoli cell by suppressing AMH production and mRNA expression of AMH, SOX9, DHH, and COL2A1. While PGE2 production was suppressed by ibuprofen, PGD2 production was not. Germ cell transcripts POU5F1, TFAP2C, LIN28A, ALPP and KIT were also reduced by ibuprofen. We conclude that, at concentrations relevant to human exposure and within a particular narrow 'early window' of sensitivity within first trimester, ibuprofen causes direct endocrine disturbances in the human fetal testis and alteration of the germ cell biology.
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http://dx.doi.org/10.1038/srep44184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345102PMC
March 2017

Mammary alveolar epithelial cells convert to brown adipocytes in post-lactating mice.

J Cell Physiol 2017 Nov 24;232(11):2923-2928. Epub 2017 Mar 24.

Department of Experimental and Clinical Medicine, University of Ancona (Università Politecnica delle Marche), Ancona, Italy.

During pregnancy and lactation, subcutaneous white adipocytes in the mouse mammary gland transdifferentiate reversibly to milk-secreting epithelial cells. In this study, we demonstrate by transmission electron microscopy that in the post-lactating mammary gland interscapular multilocular adipocytes found close to the mammary alveoli contain milk protein granules. Use of the Cre-loxP recombination system allowed showing that the involuting mammary gland of whey acidic protein-Cre/R26R mice, whose secretory alveolar cells express the lacZ gene during pregnancy, contains some X-Gal-stained and uncoupling protein 1-positive interscapular multilocular adipocytes. These data suggest that during mammary gland involution some milk-secreting epithelial cells in the anterior subcutaneous depot may transdifferentiate to brown adipocytes, highlighting a hitherto unappreciated feature of mouse adipose organ plasticity.
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http://dx.doi.org/10.1002/jcp.25858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526734PMC
November 2017

ATGC database and ATGC-COGs: an updated resource for micro- and macro-evolutionary studies of prokaryotic genomes and protein family annotation.

Nucleic Acids Res 2017 01 18;45(D1):D210-D218. Epub 2016 Oct 18.

National Center for Biotechnology Information, National Library of Medicine, National Institute of Health, Bethesda, MD 20894, USA.

The Alignable Tight Genomic Clusters (ATGCs) database is a collection of closely related bacterial and archaeal genomes that provides several tools to aid research into evolutionary processes in the microbial world. Each ATGC is a taxonomy-independent cluster of 2 or more completely sequenced genomes that meet the objective criteria of a high degree of local gene order (synteny) and a small number of synonymous substitutions in the protein-coding genes. As such, each ATGC is suited for analysis of microevolutionary variations within a cohesive group of organisms (e.g. species), whereas the entire collection of ATGCs is useful for macroevolutionary studies. The ATGC database includes many forms of pre-computed data, in particular ATGC-COGs (Clusters of Orthologous Genes), multiple sequence alignments, a set of 'index' orthologs representing the most well-conserved members of each ATGC-COG, the phylogenetic tree of the organisms within each ATGC, etc. Although the ATGC database contains several million proteins from thousands of genomes organized into hundreds of clusters (roughly a 4-fold increase since the last version of the ATGC database), it is now built with completely automated methods and will be regularly updated following new releases of the NCBI RefSeq database. The ATGC database is hosted jointly at the University of Iowa at dmk-brain.ecn.uiowa.edu/ATGC/ and the NCBI at ftp.ncbi.nlm.nih.gov/pub/kristensen/ATGC/atgc_home.html.
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http://dx.doi.org/10.1093/nar/gkw934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210634PMC
January 2017

Evolutionary switches between two serine codon sets are driven by selection.

Proc Natl Acad Sci U S A 2016 11 31;113(46):13109-13113. Epub 2016 Oct 31.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894;

Serine is the only amino acid that is encoded by two disjoint codon sets so that a tandem substitution of two nucleotides is required to switch between the two sets. Previously published evidence suggests that, for the most evolutionarily conserved serines, the codon set switch occurs by simultaneous substitution of two nucleotides. Here we report a genome-wide reconstruction of the evolution of serine codons in triplets of closely related species from diverse prokaryotes and eukaryotes. The results indicate that the great majority of codon set switches proceed by two consecutive nucleotide substitutions, via a threonine or cysteine intermediate, and are driven by selection. These findings imply a strong pressure of purifying selection in protein evolution, which in the case of serine codon set switches occurs via an initial deleterious substitution quickly followed by a second, compensatory substitution. The result is frequent reversal of amino acid replacements and, at short evolutionary distances, pervasive homoplasy.
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http://dx.doi.org/10.1073/pnas.1615832113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135333PMC
November 2016

Prokaryotic Virus Orthologous Groups (pVOGs): a resource for comparative genomics and protein family annotation.

Nucleic Acids Res 2017 01 26;45(D1):D491-D498. Epub 2016 Oct 26.

Department of Biomedical Engineering, College of Engineering, University of Iowa, Iowa City, IA 52242, USA

Viruses are the most abundant and diverse biological entities on earth, and while most of this diversity remains completely unexplored, advances in genome sequencing have provided unprecedented glimpses into the virosphere. The Prokaryotic Virus Orthologous Groups (pVOGs, formerly called Phage Orthologous Groups, POGs) resource has aided in this task over the past decade by using automated methods to keep pace with the rapid increase in genomic data. The uses of pVOGs include functional annotation of viral proteins, identification of genes and viruses in uncharacterized DNA samples, phylogenetic analysis, large-scale comparative genomics projects, and more. The pVOGs database represents a comprehensive set of orthologous gene families shared across multiple complete genomes of viruses that infect bacterial or archaeal hosts (viruses of eukaryotes will be added at a future date). The pVOGs are constructed within the Clusters of Orthologous Groups (COGs) framework that is widely used for orthology identification in prokaryotes. Since the previous release of the POGs, the size has tripled to nearly 3000 genomes and 300 000 proteins, and the number of conserved orthologous groups doubled to 9518. User-friendly webpages are available, including multiple sequence alignments and HMM profiles for each VOG. These changes provide major improvements to the pVOGs database, at a time of rapid advances in virus genomics. The pVOGs database is hosted jointly at the University of Iowa at http://dmk-brain.ecn.uiowa.edu/pVOGs and the NCBI at ftp://ftp.ncbi.nlm.nih.gov/pub/kristensen/pVOGs/home.html.
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http://dx.doi.org/10.1093/nar/gkw975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210652PMC
January 2017

Analgesic use - prevalence, biomonitoring and endocrine and reproductive effects.

Nat Rev Endocrinol 2016 07 6;12(7):381-93. Epub 2016 May 6.

Institut national de la santé et de la recherche médicale (Inserm), Institut de recherche en santé, environnement et travail (Irset-Inserm UMR 1085), 9 Avenue Léon Bernard, F-35042 RENNES, France.

Paracetamol and NSAIDs, in particular acetylsalicylic acid (aspirin) and ibuprofen, are among the most used and environmentally released pharmaceutical drugs. The differences in international trends in the sale and consumption of mild analgesics reflect differences in marketing, governmental policies, habits, accessibility, disease patterns and the age distribution of each population. Biomonitoring indicates ubiquitous and high human exposure to paracetamol and to salicylic acid, which is the main metabolite of acetylsalicylic acid. Furthermore, evidence suggests that analgesics can have endocrine disruptive properties capable of altering animal and human reproductive function from fetal life to adulthood in both sexes. Medical and public awareness about these health concerns should be increased, particularly among pregnant women.
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http://dx.doi.org/10.1038/nrendo.2016.55DOI Listing
July 2016

A census of α-helical membrane proteins in double-stranded DNA viruses infecting bacteria and archaea.

BMC Bioinformatics 2015 Nov 10;16:380. Epub 2015 Nov 10.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA.

Background: Viruses are the most abundant and genetically diverse biological entities on earth, yet the repertoire of viral proteins remains poorly explored. As the number of sequenced virus genomes grows into the thousands, and the number of viral proteins into the hundreds of thousands, we report a systematic computational analysis of the point of first-contact between viruses and their hosts, namely viral transmembrane (TM) proteins.

Results: The complement of α-helical TM proteins in double-stranded DNA viruses infecting bacteria and archaea reveals large-scale trends that differ from those of their hosts. Viruses typically encode a substantially lower fraction of TM proteins than archaea or bacteria, with the notable exception of viruses with virions containing a lipid component such as a lipid envelope, internal lipid core, or inner membrane vesicle. Compared to bacteriophages, archaeal viruses are substantially enriched in membrane proteins. However, this feature is not always stable throughout the evolution of a viral lineage; for example, TM proteins are not part of the common heritage shared between Lipothrixviridae and Rudiviridae. In contrast to bacteria and archaea, viruses almost completely lack proteins with complicated membrane topologies composed of more than 4 TM segments, with the few detected exceptions being obvious cases of relatively recent horizontal transfer from the host.

Conclusions: The dramatic differences between the membrane proteomes of cells and viruses stem from the fact that viruses do not depend on essential membranes for energy transformation, ion homeostasis, nutrient transport and signaling.
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http://dx.doi.org/10.1186/s12859-015-0817-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641393PMC
November 2015

Evolutionary Conservation of Bacterial Essential Metabolic Genes across All Bacterial Culture Media.

PLoS One 2015 20;10(4):e0123785. Epub 2015 Apr 20.

The Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, Israel; The Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; Dept. of Computer Science and the Center for Bioinformatics & Computational Biology, the University of Maryland, Maryland, United States of America.

One of the basic postulates of molecular evolution is that functionally important genes should evolve slower than genes of lesser significance. Essential genes, whose knockout leads to a lethal phenotype are considered of high functional importance, yet whether they are truly more conserved than nonessential genes has been the topic of much debate, fuelled by a host of contradictory findings. Here we conduct the first large-scale study utilizing genome-scale metabolic modeling and spanning many bacterial species, which aims to answer this question. Using the novel Media Variation Analysis, we examine the range of conservation of essential vs. nonessential metabolic genes in a given species across all possible media. We are thus able to obtain for the first time, exact upper and lower bounds on the levels of differential conservation of essential genes for each of the species studied. The results show that bacteria do exhibit an overall tendency for differential conservation of their essential genes vs. their non-essential ones, yet this tendency is highly variable across species. We show that the model bacterium E. coli K12 may or may not exhibit differential conservation of essential genes depending on its growth medium, shedding light on previous experimental studies showing opposite trends.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123785PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403854PMC
January 2016

No evidence of inhibition of horizontal gene transfer by CRISPR-Cas on evolutionary timescales.

ISME J 2015 Sep 24;9(9):2021-7. Epub 2015 Feb 24.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA.

The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)-Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR-Cas maintenance and one of the causes of the patchy distribution of CRISPR-Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR-Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR-Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution.
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http://dx.doi.org/10.1038/ismej.2015.20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4542034PMC
September 2015

Genomes in turmoil: quantification of genome dynamics in prokaryote supergenomes.

BMC Biol 2014 Aug 21;12:66. Epub 2014 Aug 21.

Background: Genomes of bacteria and archaea (collectively, prokaryotes) appear to exist in incessant flux, expanding via horizontal gene transfer and gene duplication, and contracting via gene loss. However, the actual rates of genome dynamics and relative contributions of different types of event across the diversity of prokaryotes are largely unknown, as are the sizes of microbial supergenomes, i.e. pools of genes that are accessible to the given microbial species.

Results: We performed a comprehensive analysis of the genome dynamics in 35 groups (34 bacterial and one archaeal) of closely related microbial genomes using a phylogenetic birth-and-death maximum likelihood model to quantify the rates of gene family gain and loss, as well as expansion and reduction. The results show that loss of gene families dominates the evolution of prokaryotes, occurring at approximately three times the rate of gain. The rates of gene family expansion and reduction are typically seven and twenty times less than the gain and loss rates, respectively. Thus, the prevailing mode of evolution in bacteria and archaea is genome contraction, which is partially compensated by the gain of new gene families via horizontal gene transfer. However, the rates of gene family gain, loss, expansion and reduction vary within wide ranges, with the most stable genomes showing rates about 25 times lower than the most dynamic genomes. For many groups, the supergenome estimated from the fraction of repetitive gene family gains includes about tenfold more gene families than the typical genome in the group although some groups appear to have vast, 'open' supergenomes.

Conclusions: Reconstruction of evolution for groups of closely related bacteria and archaea reveals an extremely rapid and highly variable flux of genes in evolving microbial genomes, demonstrates that extensive gene loss and horizontal gene transfer leading to innovation are the two dominant evolutionary processes, and yields robust estimates of the supergenome size.
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http://dx.doi.org/10.1186/s12915-014-0066-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166000PMC
August 2014

Indomethacin treatment prevents high fat diet-induced obesity and insulin resistance but not glucose intolerance in C57BL/6J mice.

J Biol Chem 2014 Jun 17;289(23):16032-45. Epub 2014 Apr 17.

From the Department of Biology, University of Copenhagen, 1165 Copenhagen, Denmark, the National Institute of Nutrition and Seafood Research, 5817 Bergen, Norway,

Chronic low grade inflammation is closely linked to obesity-associated insulin resistance. To examine how administration of the anti-inflammatory compound indomethacin, a general cyclooxygenase inhibitor, affected obesity development and insulin sensitivity, we fed obesity-prone male C57BL/6J mice a high fat/high sucrose (HF/HS) diet or a regular diet supplemented or not with indomethacin (±INDO) for 7 weeks. Development of obesity, insulin resistance, and glucose intolerance was monitored, and the effect of indomethacin on glucose-stimulated insulin secretion (GSIS) was measured in vivo and in vitro using MIN6 β-cells. We found that supplementation with indomethacin prevented HF/HS-induced obesity and diet-induced changes in systemic insulin sensitivity. Thus, HF/HS+INDO-fed mice remained insulin-sensitive. However, mice fed HF/HS+INDO exhibited pronounced glucose intolerance. Hepatic glucose output was significantly increased. Indomethacin had no effect on adipose tissue mass, glucose tolerance, or GSIS when included in a regular diet. Indomethacin administration to obese mice did not reduce adipose tissue mass, and the compensatory increase in GSIS observed in obese mice was not affected by treatment with indomethacin. We demonstrate that indomethacin did not inhibit GSIS per se, but activation of GPR40 in the presence of indomethacin inhibited glucose-dependent insulin secretion in MIN6 cells. We conclude that constitutive high hepatic glucose output combined with impaired GSIS in response to activation of GPR40-dependent signaling in the HF/HS+INDO-fed mice contributed to the impaired glucose clearance during a glucose challenge and that the resulting lower levels of plasma insulin prevented the obesogenic action of the HF/HS diet.
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http://dx.doi.org/10.1074/jbc.M113.525220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047378PMC
June 2014

Coupling between protein level selection and codon usage optimization in the evolution of bacteria and archaea.

mBio 2014 Mar 25;5(2):e00956-14. Epub 2014 Mar 25.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA.

The relationship between the selection affecting codon usage and selection on protein sequences of orthologous genes in diverse groups of bacteria and archaea was examined by using the Alignable Tight Genome Clusters database of prokaryote genomes. The codon usage bias is generally low, with 57.5% of the gene-specific optimal codon frequencies (Fopt) being below 0.55. This apparent weak selection on codon usage contrasts with the strong purifying selection on amino acid sequences, with 65.8% of the gene-specific dN/dS ratios being below 0.1. For most of the genomes compared, a limited but statistically significant negative correlation between Fopt and dN/dS was observed, which is indicative of a link between selection on protein sequence and selection on codon usage. The strength of the coupling between the protein level selection and codon usage bias showed a strong positive correlation with the genomic GC content. Combined with previous observations on the selection for GC-rich codons in bacteria and archaea with GC-rich genomes, these findings suggest that selection for translational fine-tuning could be an important factor in microbial evolution that drives the evolution of genome GC content away from mutational equilibrium. This type of selection is particularly pronounced in slowly evolving, "high-status" genes. A significantly stronger link between the two aspects of selection is observed in free-living bacteria than in parasitic bacteria and in genes encoding metabolic enzymes and transporters than in informational genes. These differences might reflect the special importance of translational fine-tuning for the adaptability of gene expression to environmental changes. The results of this work establish the coupling between protein level selection and selection for translational optimization as a distinct and potentially important factor in microbial evolution. IMPORTANCE Selection affects the evolution of microbial genomes at many levels, including both the structure of proteins and the regulation of their production. Here we demonstrate the coupling between the selection on protein sequences and the optimization of codon usage in a broad range of bacteria and archaea. The strength of this coupling varies over a wide range and strongly and positively correlates with the genomic GC content. The cause(s) of the evolution of high GC content is a long-standing open question, given the universal mutational bias toward AT. We propose that optimization of codon usage could be one of the key factors that determine the evolution of GC-rich genomes. This work establishes the coupling between selection at the level of protein sequence and at the level of codon choice optimization as a distinct aspect of genome evolution.
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http://dx.doi.org/10.1128/mBio.00956-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977353PMC
March 2014

Classification and quantification of bacteriophage taxa in human gut metagenomes.

ISME J 2014 Jul 13;8(7):1391-402. Epub 2014 Mar 13.

Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

Bacteriophages have key roles in microbial communities, to a large extent shaping the taxonomic and functional composition of the microbiome, but data on the connections between phage diversity and the composition of communities are scarce. Using taxon-specific marker genes, we identified and monitored 20 viral taxa in 252 human gut metagenomic samples, mostly at the level of genera. On average, five phage taxa were identified in each sample, with up to three of these being highly abundant. The abundances of most phage taxa vary by up to four orders of magnitude between the samples, and several taxa that are highly abundant in some samples are absent in others. Significant correlations exist between the abundances of some phage taxa and human host metadata: for example, 'Group 936 lactococcal phages' are more prevalent and abundant in Danish samples than in samples from Spain or the United States of America. Quantification of phages that exist as integrated prophages revealed that the abundance profiles of prophages are highly individual-specific and remain unique to an individual over a 1-year time period, and prediction of prophage lysis across the samples identified hundreds of prophages that are apparently active in the gut and vary across the samples, in terms of presence and lytic state. Finally, a prophage-host network of the human gut was established and includes numerous novel host-phage associations.
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http://dx.doi.org/10.1038/ismej.2014.30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069399PMC
July 2014

Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.

J Clin Endocrinol Metab 2013 Nov 12;98(11):E1757-67. Epub 2013 Sep 12.

Institut de Recherche en Santé, Environnement et Travail-Institut National de la Santé et de la Recherche Médicale U1085, Campus de Beaulieu, F-35042 Rennes, France.

Context: Masculinization depends on the fetal testis. Exposure of the human fetus during pregnancy to paracetamol and/or to other mild analgesics is associated with an increased risk of cryptorchidism.

Objective: We aimed to determine whether mild analgesics disrupted the morphology and endocrine function of the human testis.

Design: We used an in vitro system based on the culture of human fetal testes exposed or not to paracetamol, its metabolite N-(4-hydroxyphenyl)-arachidonoylethanolamide (AM404), aspirin, indomethacin, and ketoconazole at 10(-4) to 10(-7) M.

Setting: The study was conducted at the University of Rennes I.

Patients/participants: Human fetal testes were from pregnant women after induced abortion, between 7 and 12 weeks of gestation (GW).

Main Outcome Measures: Testosterone (RIA), anti-Müllerian hormone (ELISA), insulin-like factor 3 (RIA), and prostaglandin (PG) D2 and PGE2 (ELISA) were assayed in the medium. Testicular cells were counted using histology and image analysis. The possible nuclear receptor-mediated activities of the analgesics were investigated using reporter cell lines expressing estrogen, androgen, and peroxisome proliferator-activated γ receptors.

Results: Indomethacin and aspirin stimulated testosterone production, particularly by the younger testes (8-9 GW vs 10-12 GW). Paracetamol, AM404, and ketoconazole decreased insulin-like factor 3 levels. Aspirin stimulated whereas ketoconazole inhibited AMH production. PGE2 levels were inhibited by paracetamol and aspirin in the 7 to 12 GW testes and by indomethacin but only in 7 to 9.86 GW testes. The inhibitory trends seen for PGD2 were not statistically significant.

Conclusions: Analgesics at concentrations relevant to human exposure cause endocrine disturbances in the fetal testis. We suggest that the fetal human testis displays slight critical age windows for sensitivity to direct exposure to aspirin, indomethacin, and paracetamol. The analgesic-induced inhibition of INSL3 may be the mechanism by which analgesics increase the risk of cryptorchidism. Greater caution is required concerning consumption of analgesics during pregnancy.
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http://dx.doi.org/10.1210/jc.2013-2531DOI Listing
November 2013

Orthologous gene clusters and taxon signature genes for viruses of prokaryotes.

J Bacteriol 2013 Mar 7;195(5):941-50. Epub 2012 Dec 7.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA.

Viruses are the most abundant biological entities on earth and encompass a vast amount of genetic diversity. The recent rapid increase in the number of sequenced viral genomes has created unprecedented opportunities for gaining new insight into the structure and evolution of the virosphere. Here, we present an update of the phage orthologous groups (POGs), a collection of 4,542 clusters of orthologous genes from bacteriophages that now also includes viruses infecting archaea and encompasses more than 1,000 distinct virus genomes. Analysis of this expanded data set shows that the number of POGs keeps growing without saturation and that a substantial majority of the POGs remain specific to viruses, lacking homologues in prokaryotic cells, outside known proviruses. Thus, the great majority of virus genes apparently remains to be discovered. A complementary observation is that numerous viral genomes remain poorly, if at all, covered by POGs. The genome coverage by POGs is expected to increase as more genomes are sequenced. Taxon-specific, single-copy signature genes that are not observed in prokaryotic genomes outside detected proviruses were identified for two-thirds of the 57 taxa (those with genomes available from at least 3 distinct viruses), with half of these present in all members of the respective taxon. These signatures can be used to specifically identify the presence and quantify the abundance of viruses from particular taxa in metagenomic samples and thus gain new insights into the ecology and evolution of viruses in relation to their hosts.
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http://dx.doi.org/10.1128/JB.01801-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571318PMC
March 2013

Contribution of phage-derived genomic islands to the virulence of facultative bacterial pathogens.

Environ Microbiol 2013 Feb 4;15(2):307-12. Epub 2012 Oct 4.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.

Facultative pathogens have extremely dynamic pan-genomes, to a large extent derived from bacteriophages and other mobile elements. We developed a simple approach to identify phage-derived genomic islands and apply it to show that pathogens from diverse bacterial genera are significantly enriched in clustered phage-derived genes compared with related benign strains. These findings show that genome expansion by integration of prophages containing virulence factors is a major route of evolution of facultative bacterial pathogens.
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http://dx.doi.org/10.1111/j.1462-2920.2012.02886.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866053PMC
February 2013

Moving beyond genome sequencing into personalized genomic medicine: biological and computing challenges.

Genome Biol 2011 Oct 24;12(10):308. Epub 2011 Oct 24.

National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894, USA.

A report of the second annual Beyond the Genome conference held on the 19-22 September 2011 at The Universities at Shady Grove, Rockville, Maryland, USA, where increases in computing that may help make personal genomics a reality were a major focus.
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http://dx.doi.org/10.1186/gb-2011-12-10-308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3333770PMC
October 2011

Computational methods for Gene Orthology inference.

Brief Bioinform 2011 Sep 19;12(5):379-91. Epub 2011 Jun 19.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.

Accurate inference of orthologous genes is a pre-requisite for most comparative genomics studies, and is also important for functional annotation of new genomes. Identification of orthologous gene sets typically involves phylogenetic tree analysis, heuristic algorithms based on sequence conservation, synteny analysis, or some combination of these approaches. The most direct tree-based methods typically rely on the comparison of an individual gene tree with a species tree. Once the two trees are accurately constructed, orthologs are straightforwardly identified by the definition of orthology as those homologs that are related by speciation, rather than gene duplication, at their most recent point of origin. Although ideal for the purpose of orthology identification in principle, phylogenetic trees are computationally expensive to construct for large numbers of genes and genomes, and they often contain errors, especially at large evolutionary distances. Moreover, in many organisms, in particular prokaryotes and viruses, evolution does not appear to have followed a simple 'tree-like' mode, which makes conventional tree reconciliation inapplicable. Other, heuristic methods identify probable orthologs as the closest homologous pairs or groups of genes in a set of organisms. These approaches are faster and easier to automate than tree-based methods, with efficient implementations provided by graph-theoretical algorithms enabling comparisons of thousands of genomes. Comparisons of these two approaches show that, despite conceptual differences, they produce similar sets of orthologs, especially at short evolutionary distances. Synteny also can aid in identification of orthologs. Often, tree-based, sequence similarity- and synteny-based approaches can be combined into flexible hybrid methods.
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http://dx.doi.org/10.1093/bib/bbr030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178053PMC
September 2011