Ensembl 2023.

Ensembl (https://www.ensembl.org) has produced high-quality genomic resources for vertebrates and model organisms for more than twenty years. During that time, our resources, services and tools have continually evolved in line with both the publicly available genome data and the downstream research and applications that utilise the Ensembl platform. In recent years we have witnessed a dramatic shift in the genomic landscape. There has been a large increase in the number of high-quality reference genomes through global biodiversity initiatives. In parallel, there have been major advances towards pangenome representations of higher species, where many alternative genome assemblies representing different breeds, cultivars, strains and haplotypes are now available. In order to support these efforts and accelerate downstream research, it is our goal at Ensembl to create high-quality annotations, tools and services for species across the tree of life. Here, we report our resources for popular reference genomes, the dramatic growth of our annotations (including haplotypes from the first human pangenome graphs), updates to the Ensembl Variant Effect Predictor (VEP), interactive protein structure predictions from AlphaFold DB, and the beta release of our new website.

Amphioxus functional genomics and the origins of vertebrate gene regulation.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

Ensembl 2022.

Ensembl (https://www.ensembl.org) is unique in its flexible infrastructure for access to genomic data and annotation. It has been designed to efficiently deliver annotation at scale for all eukaryotic life, and it also provides deep comprehensive annotation for key species. Genomes representing a greater diversity of species are increasingly being sequenced. In response, we have focussed our recent efforts on expediting the annotation of new assemblies. Here, we report the release of the greatest annual number of newly annotated genomes in the history of Ensembl via our dedicated Ensembl Rapid Release platform (http://rapid.ensembl.org). We have also developed a new method to generate comparative analyses at scale for these assemblies and, for the first time, we have annotated non-vertebrate eukaryotes. Meanwhile, we continually improve, extend and update the annotation for our high-value reference vertebrate genomes and report the details here. We have a range of specific software tools for specific tasks, such as the Ensembl Variant Effect Predictor (VEP) and the newly developed interface for the Variant Recoder. All Ensembl data, software and tools are freely available for download and are accessible programmatically.

Holography from Singular Supertranslations on a Black Hole Horizon.

We investigate the standard and dual Bondi-Metzner-Sachs (BMS) supertranslation generators on a black hole horizon and draw some conclusions about black hole physics. Recently, it has been shown that in addition to conventional BMS supertranslation symmetries, there exists an additional infinite set of magnetic asymptotic symmetries, dual BMS supertranslations, again parametrized by a function on the two-sphere. We show that the Dirac bracket between these generators exhibits an anomalous central term when one parameter function exhibits a singularity in the complex stereographical coordinates on the sphere. In order to preserve general coordinate invariance, we demonstrate that this central term can be removed by postulating a holographic gravitational Chern-Simons theory on the horizon. This indicates that for an anomaly-free theory of quantum gravity in the presence of a black hole, one should include a boundary theory on the horizon.

Asymptotic Gravitational Charges.

We present a method for finding, in principle, all asymptotic gravitational charges. The basic idea is that one must consider all possible contributions to the action that do not affect the equations of motion for the theory of interest; such terms include topological terms. As a result we observe that the first order formalism is best suited to an analysis of asymptotic charges. In particular, this method can be used to provide a Hamiltonian derivation of recently found dual charges.

Soft Hair on Black Holes.

It has recently been shown that Bondi-van der Burg-Metzner-Sachs supertranslation symmetries imply an infinite number of conservation laws for all gravitational theories in asymptotically Minkowskian spacetimes. These laws require black holes to carry a large amount of soft (i.e., zero-energy) supertranslation hair. The presence of a Maxwell field similarly implies soft electric hair. This Letter gives an explicit description of soft hair in terms of soft gravitons or photons on the black hole horizon, and shows that complete information about their quantum state is stored on a holographic plate at the future boundary of the horizon. Charge conservation is used to give an infinite number of exact relations between the evaporation products of black holes which have different soft hair but are otherwise identical. It is further argued that soft hair which is spatially localized to much less than a Planck length cannot be excited in a physically realizable process, giving an effective number of soft degrees of freedom proportional to the horizon area in Planck units.

Lightest Visible-Sector Supersymmetric Particle is Likely to be Unstable.

We argue, based on typical properties of known solutions of string or M theory, that the lightest supersymmetric particle of the visible sector is likely to be unstable. In other words, dark matter is probably not a particle with standard model quantum numbers, such as a weakly interacting massive particle. The argument is simple and based on the typical occurrence of (a) hidden sectors, (b) interactions between the standard model (visible) sector and these hidden sectors, and (c) the lack of an argument against massive neutral hidden sector particles being lighter than the lightest visible supersymmetric particle. These conclusions do not rely on arguments such as R-parity violation.

GenomicInteractions: An R/Bioconductor package for manipulating and investigating chromatin interaction data.

BACKGROUND: Precise quantitative and spatiotemporal control of gene expression is necessary to ensure proper cellular differentiation and the maintenance of homeostasis. The relationship between gene expression and the spatial organisation of chromatin is highly complex, interdependent and not completely understood. The development of experimental techniques to interrogate both the higher-order structure of chromatin and the interactions between regulatory elements has recently lead to important insights on how gene expression is controlled. The ability to gain these and future insights is critically dependent on computational tools for the analysis and visualisation of data produced by these techniques. RESULTS AND CONCLUSION: We have developed GenomicInteractions, a freely available R/Bioconductor package designed for processing, analysis and visualisation of data generated from various types of chromosome conformation capture experiments. The package allows the easy annotation and summarisation of large genome-wide datasets at both the level of individual interactions and sets of genomic features, and provides several different methods for interrogating and visualising this type of data. We demonstrate this package's utility by showing example analyses performed on interaction datasets generated using Hi-C and ChIA-PET.

Structure of the O-antigen polysaccharide present in the lipopolysaccharide of Cronobacter dublinensis (subspecies lactaridi or lausannensis) HPB 3169.

Cronobacter dublinensis (formerly Enterobacter sakazakii) HPB 3169 is a pathogenic Gram-negative bacterium that produces a smooth-type lipopolysaccharide in which the antigenic O-polysaccharide component was determined to be a repeating pentasaccharide unit composed of L-rhamnose; 2-acetamido-2-deoxy-D-glucose; 3,6-dideoxy-3-(R)-3-hydroxybutyramido-D-glucose; and 3-deoxy-manno-oct-2-ulosonic acid in the respective molar ratio 2:1:1:1. Chemical and 2D NMR analyses of the O-polysaccharide and a pentasaccharide derived by the mild acid hydrolysis of the ketosyl linkage of the Kdo (3-deoxy-D-manno-2-octulosonic acid) residue in the O-polysaccharide established that the O-antigen is a high molecular mass unbranched polymer of a repeating pentasaccharide unit and has the structure [see formula in text] where Bu is a (R)-3-hydroxybutanoyl substituent. The O-antigen is structurally similar to that of the recently reported Cronobacter sakazakii strain G706 (designated as serotype O5), except that in strain G706 the d-Qui3N is in its N-acetyl form, in contrast to its presence as a 3-deoxy-3-(R)-3-hydroxybutyramido derivative in the C. sakazakii HPB 3169 strain O-antigen.

A new rapid and micro-scale hydrolysis, using triethylamine citrate, for lipopolysaccharide characterization by mass spectrometry.

Endotoxin (lipopolysaccharide, LPS) is, in general, composed of two moieties: a hydrophilic polysaccharide linked to a hydrophobic lipid A terminal unit and forms a major surface component of gram-negative bacteria. The structural features of LPS moieties play a role in pathogenesis and also involve immunogenicity and diagnostic serology. The major toxic factor of LPS resides in the lipid A moiety, anchored in the outer layer of the bacterium, and its relative biological activity is critically related to fine structural features within the molecule. In establishing relationships between structural features and biological activities of LPS it is of the utmost importance to develop new analytical methods that can be applied to the complete unambiguous characterization of a specific LPS molecule. Herein is presented a practical rapid and sensitive analytical procedure for the mass spectral screening of LPS using triethylamine citrate as an agent for both disaggregation and mild hydrolysis of LPS. It provides improved matrix-assisted laser desorption/ionization (MALDI) mass spectra and, in particular, affords the identification of fragments retaining labile substituents present in the native macromolecular LPS structures. The methods were developed and applied using purified LPS of Escherichia coli and Salmonella enterica, as well as more complex LPS of Actnobacillus pleuropneumoniae.

The structure of the antigenic O-polysaccharide in the lipopolysaccharide of enterohaemorrhagic Escherichia coli serotype O71:H12.

The antigenic O-polysaccharide component of the lipopolysaccharide produced by Escherichia coli serotype O71:H12 was analyzed by chemical composition, nuclear magnetic spectroscopy, and Smith-type periodate oxidation methods. It was determined to be a partially O-acetylated unbranched polymer of a repeating tetrasaccharide unit composed of L-rhamnose, D-galactose, 2-acetamido-2-deoxy-D-galactose, and 3-acetamido-3-deoxy-D-quinovose (1:1:1:1) residues having the following structure: [structure: see text]

Study of matrix additives for sensitive analysis of lipid A by matrix-assisted laser desorption ionization mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely used for structural characterization of bacterial endotoxins (lipid A). However, the mass spectrometric behavior of the lipid A molecule is highly dependent on the matrix. Furthermore, this dependence is strongly linked to phosphorylation patterns. Using lipid A from Escherichia coli O116 as a model system, we have investigated the effects of different matrices and comatrix compounds on the analysis of lipid A. In this paper, we report a highly sensitive matrix system for lipid A analysis, which consists of 5-chloro-2-mercaptobenzothiazole matrix and EDTA ammonium salt comatrix. This matrix system enhances the sensitivity of the analysis of diphosphorylated lipid A species by more than 100-fold and in addition provides tolerance to high concentrations of sodium dodecyl sulfate (SDS) and tolerance to sodium chloride and calcium chloride at 10 muM, 100 muM, and 10 muM concentrations. The method was further evaluated for analysis of lipid A species with different phosphorylation patterns and from different bacteria, including Helicobacter pylori, Salmonella enterica serovar Riogrande, and Francisella novicida.

Structural determination of the O-antigenic polysaccharide of enteropathogenic Escherichia coli O103:H2.

The structure of the antigenic O-polysaccharide isolated from the lipopolysaccharide produced by enterohemorrhagic Escherichia coli O103:H2 was determined and shown to be composed of d-glucose (1 part), 2-acetamido-2-deoxy-d-glucose (2 parts), 2-acetamido-2-deoxy-d-galactose (1 part), and 3-deoxy-3-(R)-3-hydroxybutyramido-d-fucose (1 part). From the results of methylation analysis, Smith-type periodate oxidation degradation studies, and the use of one- and two-dimensional (1)H and (13)C NMR spectroscopy, the O-polysaccharide antigen was found to be an unbranched polymer of a repeating pentasaccharide unit having the following structure: -->2)-Beta-d-Glcp-(1-->2)-Beta-d-Fucp3NBu-(1-->6)-alpha-d-GlcpNAc-(1-->4)-alpha-d-GalpNAc-(1-->3)-Beta-d-GlcpNAc-(1-->,where Bu is (R)-3-hydroxybutyramido.

Structural elucidation of the novel core oligosaccharide from LPS of Burkholderia cepacia serogroup O4.

Lipopolysaccharide (LPS) is an important virulence factor of Burkholderia cepacia, an opportunistic bacterial pathogen that causes life-threatening disease in cystic fibrosis patients and immunocompromised individuals. B. cepacia LPS comprises an O-specific polysaccharide covalently linked to a core oligosaccharide (OS) which in turn is attached to a lipid A moiety. The complete structure of the LPS core oligosaccharide from B. cepacia serotype O4 was investigated by detailed NMR and mass spectrometry (MS) methods. High- (HMW) and low-molecular-weight (LMW) OSs were obtained by deacylation, dephosphorylation, and reducing-end reduction of the LPS. Glycan and NMR analyses established that both OSs contain a common inner-core structure consisting of D-glucose, L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, 3-deoxy-D-manno-octulsonic acid, and D-glycero-D-talo-2-octulosonic acid. The structure of the LMW OS differed from that of the HMW OS in that it lacks a tetra-rhamnosyl GlcNAc OS extension. These structural conclusions were confirmed by tandem MS analyses of the two OS fractions as well as an OS fraction obtained by alkaline deacylation of the LPS. The location of a phosphoethanolamine substituent in the core region was determined by ESI-MS and methylation analysis of O-deacylated LPS and core OS samples. A polyclonal antibody to B. cepacia serotype O4 core OS was cross-reactive with several other serotypes indicating common structural features.

Characterization of the O-antigen in the lipopolysaccharide of Cronobacter (Enterobacter) malonaticus 3267.

Cronobacter malonaticus, a Gram-negative bacterium previously known as Enterobacter sakazakii, is an opportunistic pathogen known to cause serious infection in infants and neonates. To provide aid for the serological and chemical identification of clinical, environmental, or food isolates of this emerging pathogen, the characterization of the lipopolysaccharide (LPS) O-polysaccharide (O-PS) antigens of Cronobacter spp. is being undertaken. The structural analysis of the O-PS, obtained by hydrazinolysis of the lipopolysaccharide produced by Cronobacter malonaticus HPB 3267, was investigated by composition, methylation, and two-dimensional high-resolution nuclear magnetic resonance methods, and was found to be a polymer of a branched pentasaccharide unit. This unit is composed of D-glucose (D-Glc), D-galactose (D-Gal), 2-amino-2-deoxy-D-glucose (D-GlcN), 2-amino-2-deoxy-D-galactose (D-GalN) and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residues (1: 1: 1: 1: 1), forms the repeating oligosaccharide in the O-PS antigen, and has the structure: [structure: see text].

Physician medical direction and clinical performance at an established emergency medical services system.

OBJECTIVE: Few developed emergency medical services (EMS) systems operate without dedicated medical direction. We describe the experience of Hamad Medical Corporation (HMC) EMS, which in 2007 first engaged an EMS medical director to develop and implement medical direction and quality assurance programs. We report subsequent changes to system performance over time. METHODS: Over one year, changes to the service's clinical infrastructure were made: Policies were revised, paramedic scopes of practice were adjusted, evidence-based clinical protocols were developed, and skills maintenance and education programs were implemented. Credentialing, physician chart auditing, clinical remediation, and online medical command/hospital notification systems were introduced. RESULTS: Following these interventions, we report associated improvements to key indicators: Chart reviews revealed significant improvements in clinical quality. A comparison of pre- and post-intervention audited charts reveals a decrease in cases requiring remediation (11% to 5%, odds ratio [OR] 0.43 [95% confidence interval (CI) 0.20-0.85], p = 0.01). The proportion of charts rated as clinically acceptable rose from 48% to 84% (OR 6 [95% CI 3.9-9.1], p < 0.001). The proportion of misplaced endotracheal tubes fell (3.8% baseline to 0.6%, OR 0.16 [95% CI 0.004-1.06], (exact) p = 0.05), corresponding to improved adherence to an airway placement policy mandating use of airway confirmation devices and securing devices (0.7% compliance to 98%, OR 714 [95% CI 64-29,334], (exact) p < 0.001). Intravenous catheter insertion in unstable cases increased from 67% of cases to 92% (OR 1.31 [95% CI 1.09-1.71], p = 0.004). EMS administration of aspirin to patients with suspected ischemic chest pain improved from 2% to 77% (OR 178 [95% CI 35-1,604], p < 0.001). CONCLUSIONS: We suggest that implementation of a physician medical direction is associated with improved clinical indicators and overall quality of care at an established EMS system.

The structure of the exocellular polysaccharide produced by Rhodococcus sp. RHA1.

Rhodococcus sp. RHA1 is a Gram-positive actinomycete capable of metabolizing a wide spectrum of organic compounds whose survival in chemically hostile environments is believed to be in part due to the production of an exocellular polysaccharide (EPS). In order to investigate the functional nature of the EPS, its structure was determined using a combinatory approach including hydrolysis, composition, and methylation, analysis methods, as well as 2D (1)H and (13)C NMR spectroscopy. The EPS was found to be a high-molecular-mass polymer of a repeating tetrasaccharide unit composed of D-glucuronic acid, D-glucose, D-galactose, L-fucose and O-acetyl (1:1:1:1:1), and has the structure:

The structure of the carbohydrate backbone of the LPS from Myxococcus xanthus strain DK1622.

Gram-negative rod shaped bacterium Myxococcus xanthus DK1622 produces a smooth-type LPS. The structure of the polysaccharide O-chain and the core-lipid A region of the LPS has been determined by chemical and spectroscopic methods. The O-chain was built up of disaccharide repeating units having the following structure: -->6)-alpha-D-Glcp-(1-->4)-alpha-D-GalpNAc6oMe*-(1--> with partially methylated GalNAc residue. The core region consisted of a phosphorylated hexasaccharide, containing one Kdo residue, unsubstituted at O-4, and no heptose residues. The lipid A component consisted of beta-GlcN-(1-->6)-alpha-GlcN1P disaccharide, N-acylated with 13-methyl-C14-3OH (iso-C15-3OH), C16-3OH, and 15-methyl-C16-3OH (iso-C17-3OH) acids. The lipid portion contained O-linked iso-C16 acid.

Topologically associating domains are ancient features that coincide with Metazoan clusters of extreme noncoding conservation.

Developmental genes in metazoan genomes are surrounded by dense clusters of conserved noncoding elements (CNEs). CNEs exhibit unexplained extreme levels of sequence conservation, with many acting as developmental long-range enhancers. Clusters of CNEs define the span of regulatory inputs for many important developmental regulators and have been described previously as genomic regulatory blocks (GRBs). Their function and distribution around important regulatory genes raises the question of how they relate to 3D conformation of these loci. Here, we show that clusters of CNEs strongly coincide with topological organisation, predicting the boundaries of hundreds of topologically associating domains (TADs) in human and Drosophila. The set of TADs that are associated with high levels of noncoding conservation exhibit distinct properties compared to TADs devoid of extreme noncoding conservation. The close correspondence between extreme noncoding conservation and TADs suggests that these TADs are ancient, revealing a regulatory architecture conserved over hundreds of millions of years.Metazoan genomes contain many clusters of conserved noncoding elements. Here, the authors provide evidence that these clusters coincide with distinct topologically associating domains in humans and Drosophila, revealing a conserved regulatory genomic architecture.

Characterization of the lipopolysaccharide and beta-glucan of the fish pathogen Francisella victoria.

Lipopolysaccharide (LPS) and beta-glucan from Francisella victoria, a fish pathogen and close relative of highly virulent mammal pathogen Francisella tularensis, have been analyzed using chemical and spectroscopy methods. The polysaccharide part of the LPS was found to contain a nonrepetitive sequence of 20 monosaccharides as well as alanine, 3-aminobutyric acid, and a novel branched amino acid, thus confirming F. victoria as a unique species. The structure identified composes the largest oligosaccharide elucidated by NMR so far, and was possible to solve using high field NMR with cold probe technology combined with the latest pulse sequences, including the first application of H2BC sequence to oligosaccharides. The non-phosphorylated lipid A region of the LPS was identical to that of other Francisellae, although one of the lipid A components has not been found in Francisella novicida. The heptoseless core-lipid A region of the LPS contained a linear pentasaccharide fragment identical to the corresponding part of F. tularensis and F. novicida LPSs, differing in side-chain substituents. The linkage region of the O-chain also closely resembled that of other Francisella. LPS preparation contained two characteristic glucans, previously observed as components of LPS preparations from other strains of Francisella: amylose and the unusual beta-(1-6)-glucan with (glycerol)2phosphate at the reducing end.