Expression and in vitro phosphorylation of the yeast transcriptional activator ADR1

The ADR1 protein activates transcription of the gene coding for the glucose repressible alcohol dehydrogenase II (ADH2) in Saccharomyces cerevisiae. Previous characterization of ADR1\sp{\rm c} mutations, which allow ADH2 to escape glucose repression, led to the hypothesis that ADR1 is inactivated post-translationally by a cAMP-dependent phosphorylation mechanism. This hypothesis was tested by investigating the in vitro phosphorylation of ADR1 and ADR1\sp{\rm c} proteins in which ADR1\sp{\rm c} proteins were predicted to display a diminished level of phosphorylation. A fragment of the ADR1 gene coding for the N-terminal 658 amino acids was fused to the lacZ gene of E. coli to allow for identification and assay of the ADR1 protein. The fused gene was placed under the control of the E. coli tac promotor to ensure efficient production. Beta-galactosidase activities in plasmid-bearing E. coli indicated that ADR1/$\beta$-galactosidase fusion proteins were being expressed. SDS-polyacrylamide gel analysis of E. coli extracts revealed discrete plasmid-encoded fusion proteins. Fusion proteins contained in crude E. coli extracts were phosphorylated in vitro using purified catalytic subunit of mammalian cAMP-dependent protein kinase (cAPK). Peptide mapping results indicated that ADR1 was phosphorylated at two discrete sites in vitro. The primary phosphorylation site was identified as serine-230. A secondary site to the N-terminal side of serine-230 was also identified. ADR1 DNA sequences were replaced in the expression plasmids with three sequences containing ADR1\sp{\rm c} mutations, each of which encodes a single amino acid substitution within the phosphorylation sequence located between residues 227-231 (Arg-Arg-Ala-Ser-Phe). The in vitro phosphorylation of ADR1 and ADR1\sp{\rm c} containing fusion proteins were compared using both bovine cAPK and the yeast cAPK catalytic subunit encoded by the TPK1 gene. Addition of purified yeast regulatory subunit completely blocked fusion protein phosphorylation by the yeast kinase in the absence of cAMP. The ADR1-2\sp{\rm c} and ADR1-5\sp{\rm c} mutations were found to diminish phosphorylation at serine-230. The ADR1-7\sp{\rm c} mutation, which substitutes a leucine residue for serine-230, completely eliminated phosphorylation. The results of this work support the hypotheses that the ADR1\sp{\rm c} proteins bypass glucose repression by avoiding phosphorylation and suggest that ADR1 is regulated by a cAMP-dependent phosphorylation. Possible mechanisms by which phosphorylation may regulate ADR1 activity are discussed

Alliance of Genome Resources Portal: unified model organism research platform

The Alliance of Genome Resources (Alliance) is a consortium of the major model organism databases and the Gene Ontology that is guided by the vision of facilitating exploration of related genes in human and well-studied model organisms by providing a highly integrated and comprehensive platform that enables researchers to leverage the extensive body of genetic and genomic studies in these organisms. Initiated in 2016, the Alliance is building a central portal (www.alliancegenome.org) for access to data for the primary model organisms along with gene ontology data and human data. All data types represented in the Alliance portal (e.g. genomic data and phenotype descriptions) have common data models and workflows for curation. All data are open and freely available via a variety of mechanisms. Long-term plans for the Alliance project include a focus on coverage of additional model organisms including those without dedicated curation communities, and the inclusion of new data types with a particular focus on providing data and tools for the non-model-organism researcher that support enhanced discovery about human health and disease. Here we review current progress and present immediate plans for this new bioinformatics resource

Alliance of Genome Resources Portal: unified model organism research platform

The Alliance of Genome Resources (Alliance) is a consortium of the major model organism databases and the Gene Ontology that is guided by the vision of facilitating exploration of related genes in human and well-studied model organisms by providing a highly integrated and comprehensive platform that enables researchers to leverage the extensive body of genetic and genomic studies in these organisms. Initiated in 2016, the Alliance is building a central portal (www.alliancegenome.org) for access to data for the primary model organisms along with gene ontology data and human data. All data types represented in the Alliance portal (e.g. genomic data and phenotype descriptions) have common data models and workflows for curation. All data are open and freely available via a variety of mechanisms. Long-term plans for the Alliance project include a focus on coverage of additional model organisms including those without dedicated curation communities, and the inclusion of new data types with a particular focus on providing data and tools for the non-model-organism researcher that support enhanced discovery about human health and disease. Here we review current progress and present immediate plans for this new bioinformatics resource

Expanded encyclopaedias of DNA elements in the human and mouse genomes

All data are available on the ENCODE data portal: www.encodeproject. org. All code is available on GitHub from the links provided in the methods section. Code related to the Registry of cCREs can be found at https:// github.com/weng-lab/ENCODE-cCREs. Code related to SCREEN can be found at https://github.com/weng-lab/SCREEN.© The Author(s) 2020. The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.This work was supported by grants from the NIH under U01HG007019, U01HG007033, U01HG007036, U01HG007037, U41HG006992, U41HG006993, U41HG006994, U41HG006995, U41HG006996, U41HG006997, U41HG006998, U41HG006999, U41HG007000, U41HG007001, U41HG007002, U41HG007003, U54HG006991, U54HG006997, U54HG006998, U54HG007004, U54HG007005, U54HG007010 and UM1HG009442

Virology under the microscope—a call for rational discourse

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns – conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we – a broad group of working virologists – seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Monolithic light concentration by core-shell TiO2 nanostructures templated by monodisperse polymer colloidal monolayers

Nanostructured dielectric overlayers can be used to increase light absorption in nanometer-thin films used for various optoelectronic applications. Here, the self-assembly of a close-packed monolayer of polystyrene nanospheres is used to template a core-shell polystyrene-TiO2 light-concentrating monolithic structure. This is enabled by the growth of TiO2 at temperatures below the polystyrene glass-transition temperature via atomic layer deposition. The result is a monolithic, tailorable nanostructured overlayer fabricated by simple chemical methods. The design of this monolith can be tailored to generate significant absorption increases in thin film light absorbers. Finite-difference, time domain simulations are used to design polystyrene-TiO2 core-shell monoliths that maximize light absorption in a 40 nm GaAs-on-Si substrate as a model for a photoconductive antenna THz emitter. An optimized core-shell monolith structure generated a greater than 60-fold increase of light absorption at a single wavelength in the GaAs layer of the model device