The Ensembl Genome Browser: Strategies for Accessing Eukaryotic Genome Data.

The Ensembl Genome Browser provides a wealth of freely available genomic data that can be accessed for many purposes by genetics, genomics, and molecular biology researchers. Herein we present two protocols for exploring different aspects of these data: a phenotype and its associated variants and genes, and a promoter and the epigenetic marks and protein-binding activity associated with it. These workflows illustrate a subset of the data types available through the Ensembl Browser, and can be considered a springboard for further exploration.

Ensembl Genomes 2018: an integrated omics infrastructure for non-vertebrate species.

Ensembl Genomes (http://www.ensemblgenomes.org) is an integrating resource for genome-scale data from non-vertebrate species, complementing the resources for vertebrate genomics developed in the Ensembl project (http://www.ensembl.org). Together, the two resources provide a consistent set of programmatic and interactive interfaces to a rich range of data including genome sequence, gene models, transcript sequence, genetic variation, and comparative analysis. This paper provides an update to the previous publications about the resource, with a focus on recent developments and expansions. These include the incorporation of almost 20 000 additional genome sequences and over 35 000 tracks of RNA-Seq data, which have been aligned to genomic sequence and made available for visualization. Other advances since 2015 include the release of the database in Resource Description Framework (RDF) format, a large increase in community-derived curation, a new high-performance protein sequence search, additional cross-references, improved annotation of non-protein-coding genes, and the launch of pre-release and archival sites. Collectively, these changes are part of a continuing response to the increasing quantity of publicly-available genome-scale data, and the consequent need to archive, integrate, annotate and disseminate these using automated, scalable methods.

Ensembl 2018.

The Ensembl project has been aggregating, processing, integrating and redistributing genomic datasets since the initial releases of the draft human genome, with the aim of accelerating genomics research through rapid open distribution of public data. Large amounts of raw data are thus transformed into knowledge, which is made available via a multitude of channels, in particular our browser (http://www.ensembl.org). Over time, we have expanded in multiple directions. First, our resources describe multiple fields of genomics, in particular gene annotation, comparative genomics, genetics and epigenomics. Second, we cover a growing number of genome assemblies; Ensembl Release 90 contains exactly 100. Third, our databases feed simultaneously into an array of services designed around different use cases, ranging from quick browsing to genome-wide bioinformatic analysis. We present here the latest developments of the Ensembl project, with a focus on managing an increasing number of assemblies, supporting efforts in genome interpretation and improving our browser.

Ensembl 2017.

Ensembl (www.ensembl.org) is a database and genome browser for enabling research on vertebrate genomes. We import, analyse, curate and integrate a diverse collection of large-scale reference data to create a more comprehensive view of genome biology than would be possible from any individual dataset. Our extensive data resources include evidence-based gene and regulatory region annotation, genome variation and gene trees. An accompanying suite of tools, infrastructure and programmatic access methods ensure uniform data analysis and distribution for all supported species. Together, these provide a comprehensive solution for large-scale and targeted genomics applications alike. Among many other developments over the past year, we have improved our resources for gene regulation and comparative genomics, and added CRISPR/Cas9 target sites. We released new browser functionality and tools, including improved filtering and prioritization of genome variation, Manhattan plot visualization for linkage disequilibrium and eQTL data, and an ontology search for phenotypes, traits and disease. We have also enhanced data discovery and access with a track hub registry and a selection of new REST end points. All Ensembl data are freely released to the scientific community and our source code is available via the open source Apache 2.0 license.

Medical countermeasures for unwanted CBRN exposures: part II radiological and nuclear threats with review of recent countermeasure patents.

INTRODUCTION: The global threat of a chemical, biological, radiological, or nuclear (CBRN) disaster is an important priority for all government agencies involved in domestic security and public health preparedness. Radiological/nuclear (RN) attacks or accidents have become a larger focus of the United States Food and Drug administration (US FDA) over time because of their increased likeliness. Clinical signs and symptoms of a developing acute radiation syndrome (ARS) are grouped into three sub-syndromes named for the dominant organ system affected, namely the hematopoietic (H-ARS), gastrointestinal (GI-ARS), and neurovascular systems. The availability of safe and effective countermeasures against radiological/nuclear threats currently represents a significant unmet medical need. Areas covered: This article reviews the development of RN threat medical countermeasures and highlights those specific countermeasures that have been recently patented and approved following the FDA Animal Rule. Patents for such agents from 2015 have been presented. Expert opinion: Two granulocyte colony-stimulating factor (G-CSF)-based radiation countermeasures (Neupogen® (Amgen, Thousand Oaks, CA) and Neulasta® (Amgen, Thousand Oaks, CA)) have recently been approved by the FDA for treatment of H-ARS and both these agents are radiomitigators, used after radiation exposure. To date, there are no FDA-approved radioprotectors for ARS.

THE POTENTIATION OF THE RADIOPROTECTIVE EFFICACY OF TWO MEDICAL COUNTERMEASURES, GAMMA-TOCOTRIENOL AND AMIFOSTINE, BY A COMBINATION PROPHYLACTIC MODALITY.

This study was designed to evaluate the possible potentiation of survival protection afforded by relatively low-dose amifostine prophylaxis against total body irradiation in combination with a protective, less toxic agent, gamma-tocotrienol (GT3). Mice were administered amifostine and/or GT3, then exposed to 9.2 Gy 60Co γ-irradiation and monitored for survival for 30 days. To investigate cytokine stimulation, mice were administered amifostine or GT3; serum samples were collected and analyzed for cytokines. Survival studies show single treatments of GT3 or amifostine significantly improved survival, compared to the vehicle, and combination treatments resulted in significantly higher survival compared to single treatments. In vivo studies with GT3 confirmed prior work indicating GT3 induces granulocyte colony-stimulating factor (G-CSF). This approach, the prophylactic combination of amifostine and GT3, which act through different mechanisms, shows promise and should be investigated further as a potential countermeasure for acute radiation syndrome.

Progenitor Cell Mobilization by Gamma-tocotrienol: A Promising Radiation Countermeasure.

This article reviews studies of progenitor mobilization with gamma-tocotrienol (GT3), a tocol under advanced development as a radiation countermeasure for acute radiation syndrome (ARS). GT3 protects mice against high doses of ionizing radiation and induces high levels of granulocyte colony-stimulating factor (G-CSF). GT3-induced G-CSF in conjunction with AMD3100 (a chemokine receptor antagonist clinically used to improve the yield of mobilized progenitors) mobilizes progenitors; these mobilized progenitors mitigate injury when infused to mice exposed to acute, high-dose ionizing radiation. The administration of a G-CSF antibody to GT3-injected donor mice abrogated the radiomitigative efficacy of blood or peripheral blood mononuclear cells (PBMC) in irradiated recipient mice. The efficacy of GT3-injected donor mice blood or PBMC was comparable to a recently published article involving blood or mononuclear cells obtained from mice injected with G-CSF. The injected progenitors were found to localize in various tissues of irradiated hosts. The authors demonstrate the efficacy of a bridging therapy in a preclinical animal model that allows the lymphohematopoietic system of severely immunocompromised mice to recover. This suggests that GT3 is a highly effective agent for radioprotection and mobilizing progenitors with significant therapeutic potential. Therefore, GT3 may be considered for further translational development and ultimately for use in humans.

Radioprotective Efficacy of Gamma-Tocotrienol in Nonhuman Primates.

The search for treatments to counter potentially lethal radiation-induced injury over the past several decades has led to the development of multiple classes of radiation countermeasures. However, to date only granulocyte colony-stimulating factor (G-CSF; filgrastim, Neupogen)and pegylated G-CSF (pegfilgrastim, Neulasta) have been approved by the United States Food and Drug Administration (FDA) for the treatment of hematopoietic acute radiation syndrome (ARS). Gamma-tocotrienol (GT3) has demonstrated strong radioprotective efficacy in the mouse model, indicating the need for further evaluation in a large animal model. In this study, we evaluated GT3 pharmacokinetics (PK) and efficacy at different doses of cobalt-60 gamma radiation (0.6 Gy/min) using the nonhuman primate (NHP) model. The PK results demonstrated increased area under the curve with increasing drug dose and half-life of GT3. GT3 treatment resulted in reduced group mean neutropenia by 3-5 days and thrombocytopenia by 1-5 days. At 5.8 and 6.5 Gy total-body irradiation, GT3 treatment completely prevented thrombocytopenia. The capability of GT3 to reduce severity and duration of neutropenia and thrombocytopenia was dose dependent; 75 mg/kg treatment was more effective than 37.5 mg/kg treatment after a 5.8 Gy dose. However, the higher GT3 dose (75 mg/kg) was associated with higher frequency of adverse skin effects (small abscess) at the injection site. GT3 treatment of irradiated NHPs caused no significant difference in animal survival at 60 days postirradiation, however, low mortality was observed in irradiated, vehicle-treated groups as well. The data from this pilot study further elucidate the role and pharmacokinetics of GT3 in hematopoietic recovery after irradiation in a NHP model, and demonstrate the potential of GT3 as a promising radioprotector.

Use of biomarkers for assessing radiation injury and efficacy of countermeasures.

Several candidate drugs for acute radiation syndrome (ARS) have been identified that have low toxicity and significant radioprotective and radiomitigative efficacy. Inasmuch as exposing healthy human volunteers to injurious levels of radiation is unethical, development and approval of new radiation countermeasures for ARS are therefore presently based on animal studies and Phase I safety study in healthy volunteers. The Animal Efficacy Rule, which underlies the Food and Drug Administration approval pathway, requires a sound understanding of the mechanisms of injury, drug efficacy, and efficacy biomarkers. In this context, it is important to identify biomarkers for radiation injury and drug efficacy that can extrapolate animal efficacy results, and can be used to convert drug doses deduced from animal studies to those that can be efficacious when used in humans. Here, we summarize the progress of studies to identify candidate biomarkers for the extent of radiation injury and for evaluation of countermeasure efficacy.

Combined immunomodulator and antimicrobial therapy eliminates polymicrobial sepsis and modulates cytokine production in combined injured mice.

PURPOSE: A combination therapy for combined injury (CI) using a non-specific immunomodulator, synthetic trehalose dicorynomycolate and monophosphoryl lipid A (STDCM-MPL), was evaluated to augment oral antimicrobial agents, levofloxacin (LVX) and amoxicillin (AMX), to eliminate endogenous sepsis and modulate cytokine production. MATERIALS AND METHODS: Female B6D2F(1)/J mice received 9.75 Gy cobalt-60 gamma-radiation and wound. Bacteria were isolated and identified in three tissues. Incidence of bacteria and cytokines were compared between treatment groups. RESULTS: Results demonstrated that the lethal dose for 50% at 30 days (LD(50/30)) of B6D2F(1)/J mice was 9.42 Gy. Antimicrobial therapy increased survival in radiation-injured (RI) mice. Combination therapy increased survival after RI and extended survival time but did not increase survival after CI. Sepsis began five days earlier in CI mice than RI mice with Gram-negative species predominating early and Gram-positive species increasing later. LVX plus AMX eliminated sepsis in CI and RI mice. STDCM-MPL eliminated Gram-positive bacteria in CI and most RI mice but not Gram-negative. Treatments significantly modulated 12 cytokines tested, which pertain to wound healing or elimination of infection. CONCLUSIONS: Combination therapy eliminates infection and prolongs survival time but does not assure CI mouse survival, suggesting that additional treatment for proliferative-cell recovery is required.

Animal models for acute radiation syndrome drug discovery.

INTRODUCTION: Although significant scientific advances have been made over the past six decades in developing safe, nontoxic and effective radiation/medical countermeasures (MCMs) for acute radiation syndrome (ARS), no drug has been approved by the US FDA. The availability of adequate animal models is a prime requisite under the criteria established by the FDA 'animal rule' for the development of novel MCMs for ARS and the discovery of biomarkers for radiation exposure. AREAS COVERED: This article reviews the developments of MCMs to combat ARS, with particular reference to the various animal models (rodents: mouse and rat; canine: beagle; minipigs and nonhuman primates [NHPs]) utilized for the in-depth evaluation. The objective, pathways and challenges of the FDA Animal Efficacy Rule are also discussed. EXPERT OPINION: There are a number of well-defined animal models, the mouse, canine and NHP, that are being used for the development of MCMs. Additional animal models, such as the minipig, are under development to further assist in the identification, efficacy testing and approval of MCMs under the FDA Animal Efficacy Rule.

Biologics as countermeasures for acute radiation syndrome: where are we now?

Despite significant scientific advances toward the development of a safe, nontoxic and effective radiation countermeasure for acute radiation syndrome (ARS) over the past six decades, no drug has been approved by the US FDA. Several biologics are currently under development as radiation countermeasures for ARS, of which three have received FDA Investigational New Drug (IND) status for clinical investigation. Presently, two of these agents, entolimod (CBLB502) and HemaMax (recombinant human IL-12) are progressing with large animal studies and clinical trials. Neupogen (G-CSF, filgrastim) has recently been recommended for approval by an FDA Advisory Committee. Filgrastim, GM-CSF (Leukine, sargramostim), and PEGylated G-CSF (Neulasta) have high potential and well-documented therapeutic effects in countering myelosuppression and may receive full licensing approval by the FDA in the future. The former two biologics are available in the US Strategic National Stockpile (SNS) for use in the event of nuclear or radiological emergency. The Emergency Use Authorization (EAU) application for entolimod may be filed soon with the FDA. Biologics are attractive agents that are progressing along the path for FDA approval, to fill the unmet need for ARS countermeasures.

Colony-stimulating factors for the treatment of the hematopoietic component of the acute radiation syndrome (H-ARS): a review.

One of the greatest national security threats to the United States is the detonation of an improvised nuclear device or a radiological dispersal device in a heavily populated area. As such, this type of security threat is considered to be of relatively low risk, but one that would have an extraordinary high impact on health and well-being of the US citizenry. Psychological counseling and medical assessments would be necessary for all those significantly impacted by the nuclear/radiological event. Direct medical interventions would be necessary for all those individuals who had received substantial radiation exposures (e.g., >1 Gy). Although no drugs or products have yet been specifically approved by the United States Food and Drug Administration (US FDA) to treat the effects of acute radiation syndrome (ARS), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and pegylated G-CSF have been used off label for treating radiation accident victims. Recent threats of terrorist attacks using nuclear or radiologic devices makes it imperative that the medical community have up-to-date information and a clear understanding of treatment protocols using therapeutically effective recombinant growth factors and cytokines such as G-CSF and GM-CSF for patients exposed to injurious doses of ionizing radiation. Based on limited human studies with underlying biology, we see that the recombinants, G-CSF and GM-CSF appear to have modest, but significant medicinal value in treating radiation accident victims. In the near future, the US FDA may approve G-CSF and GM-CSF as 'Emergency Use Authorization' (EUA) for managing radiation-induced aplasia, an ARS-related pathology. In this article, we review the status of growth factors for the treatment of radiological/nuclear accident victims.

Progenitors mobilized by gamma-tocotrienol as an effective radiation countermeasure.

The purpose of this study was to elucidate the role of gamma-tocotrienol (GT3)-mobilized progenitors in mitigating damage to mice exposed to a supralethal dose of cobalt-60 gamma-radiation. CD2F1 mice were transfused 24 h post-irradiation with whole blood or isolated peripheral blood mononuclear cells (PBMC) from donors that had received GT3 72 h prior to blood collection and recipient mice were monitored for 30 days. To understand the role of GT3-induced granulocyte colony-stimulating factor (G-CSF) in mobilizing progenitors, donor mice were administered a neutralizing antibody specific to G-CSF or its isotype before blood collection. Bacterial translocation from gut to heart, spleen and liver of irradiated recipient mice was evaluated by bacterial culture on enriched and selective agar media. Endotoxin in serum samples also was measured. We also analyzed the colony-forming units in the spleens of irradiated mice. Our results demonstrate that whole blood or PBMC from GT3-administered mice mitigated radiation injury when administered 24 h post-irradiation. Furthermore, administration of a G-CSF antibody to GT3-injected mice abrogated the efficacy of blood or PBMC obtained from such donors. Additionally, GT3-mobilized PBMC inhibited the translocation of intestinal bacteria to the heart, spleen, and liver, and increased colony forming unit-spleen (CFU-S) numbers in irradiated mice. Our data suggests that GT3 induces G-CSF, which mobilizes progenitors and these progenitors mitigate radiation injury in recipient mice. This approach using mobilized progenitor cells from GT3-injected donors could be a potential treatment for humans exposed to high doses of radiation.

Radiation countermeasure agents: an update (2011-2014).

INTRODUCTION: Despite significant scientific advances over the past 60 years towards the development of a safe, nontoxic and effective radiation countermeasure for the acute radiation syndrome (ARS), no drug has been approved by the US FDA. A radiation countermeasure to protect the population at large from the effects of lethal radiation exposure remains a significant unmet medical need of the US citizenry and, thus, has been recognized as a high priority area by the government. AREA COVERED: This article reviews relevant publications and patents for recent developments and progress for potential ARS treatments in the area of radiation countermeasures. Emphasis is placed on the advanced development of existing agents since 2011 and new agents identified as radiation countermeasure for ARS during this period. EXPERT OPINION: A number of promising radiation countermeasures are currently under development, seven of which have received US FDA investigational new drug status for clinical investigation. Four of these agents, CBLB502, Ex-RAD, HemaMax and OrbeShield, are progressing with large animal studies and clinical trials. G-CSF has high potential and well-documented therapeutic effects in countering myelosuppression and may receive full licensing approval by the US FDA in the future.

Tocols induce G-CSF and mobilise progenitors that mitigate radiation injury.

Tocols induce high levels of granulocyte-colony-stimulating factor (G-CSF). G-CSF mobilises progenitors that allow mice that have been severely immunocompromised by exposure to acute, high-dose ionising irradiation to recover and to survive. The neutralisation of G-CSF abrogates the radioprotective efficacy of tocols. This article reviews studies in which CD2F1 mice were irradiated with sufficiently high doses to cause acute radiation syndrome symptoms and then administered (iv) progenitor-enriched whole blood or peripheral blood mononuclear cells from tocol- and AMD3100-injected donor mice (AMD3100 is a chemokine receptor antagonist used to improve the yield of mobilised progenitors). In some experiments, G-CSF was neutralised completely. Irradiated recipient mice were observed for 30 d post-irradiation for survival, a primary endpoint used for determining therapeutic effectiveness. Additionally, potential tocol-induced biomarkers (cytokines, chemokines and growth factors) were quantified. The authors suggest that tocols are highly effective agents for mobilising progenitors with significant therapeutic potential.

Radioprotective efficacy of delta-tocotrienol, a vitamin E isoform, is mediated through granulocyte colony-stimulating factor.

AIMS: The objectives of this study were to determine the cytokine induction by delta tocotrienol (DT3, a promising radiation countermeasure) and to investigate the role of granulocyte colony-stimulating factor (G-CSF) in its radioprotective efficacy against ionizing radiation in mice. MAIN METHODS: Multiplex Luminex was used to analyze cytokines induced by DT3 and other tocols (gamma-tocotrienol and tocopherol succinate) in CD2F1 mice. Mice were injected with an optimal dose of DT3 and a G-CSF antibody, and their 30-day survival against cobalt-60 gamma-irradiation was monitored. The neutralization of G-CSF by the administration of a G-CSF-specific antibody in DT3-injected mice was investigated by multiplex Luminex. KEY FINDINGS: Our data demonstrate that DT3 induced high levels of various cytokines comparable to other tocols being developed as radiation countermeasures. DT3 significantly protected mice against ionizing radiation, and the administration of a G-CSF neutralizing antibody to DT3-treated animals resulted in the complete abrogation of DT3's radioprotective efficacy and neutralization of G-CSF in peripheral blood. SIGNIFICANCE: Our study findings suggest that G-CSF induced by DT3 mediates its radioprotective efficacy against ionizing radiation in mice.

Maternal dietary intake during pregnancy has longstanding consequences for the health of her offspring.

Over the past 100 years, advances in pharmaceutical and medical technology have reduced the burden of communicable disease, and our appreciation of the mechanisms underlying the development of noncommunicable disease has broadened. During this time, a number of studies, both in humans and animal models, have highlighted the importance of maintaining an optimal diet during pregnancy. In particular, a number of studies support the hypothesis that suboptimal maternal protein and fat intake during pregnancy can have long-term effects on the growing fetus, and increase the likelihood of these offspring developing cardiovascular, renal, or metabolic diseases in adulthood. More recently, it has been shown that dietary intake of a number of micronutrients may offset or reverse the deleterious effects of macronutrient imbalance. Furthermore, maternal fat intake has also been identified as a major contributor to a healthy fetal environment, with a beneficial role for unsaturated fats during development as well as a beneficial impact on cell membrane physiology. Together these studies indicate that attempts to optimise maternal nutrition may prove to be an efficient and cost-effective strategy for preventing the development of cardiovascular, renal, or metabolic diseases.

The transcription factor Eyes absent is a protein tyrosine phosphatase.

Post-translational modifications provide sensitive and flexible mechanisms to dynamically modulate protein function in response to specific signalling inputs. In the case of transcription factors, changes in phosphorylation state can influence protein stability, conformation, subcellular localization, cofactor interactions, transactivation potential and transcriptional output. Here we show that the evolutionarily conserved transcription factor Eyes absent (Eya) belongs to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily, and propose a function for it as a non-thiol-based protein tyrosine phosphatase. Experiments performed in cultured Drosophila cells and in vitro indicate that Eyes absent has intrinsic protein tyrosine phosphatase activity and can autocatalytically dephosphorylate itself. Confirming the biological significance of this function, mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. Given the functional importance of phosphorylation-dependent modulation of transcription factor activity, this evidence for a nuclear transcriptional coactivator with intrinsic phosphatase activity suggests an unanticipated method of fine-tuning transcriptional regulation.