Genomic Phenotyping by Barcode Sequencing Broadly Distinguishes between Alkylating Agents, Oxidizing Agents, and Non-Genotoxic Agents, and Reveals a Role for Aromatic Amino Acids in Cellular Recovery after Quinone Exposure

Toxicity screening of compounds provides a means to identify compounds harmful for human health and the environment. Here, we further develop the technique of genomic phenotyping to improve throughput while maintaining specificity. We exposed cells to eight different compounds that rely on different modes of action: four genotoxic alkylating (methyl methanesulfonate (MMS), N-Methyl-N-nitrosourea (MNU), N,N′-bis(2-chloroethyl)-N-nitroso-urea (BCNU), N-ethylnitrosourea (ENU)), two oxidizing (2-methylnaphthalene-1,4-dione (menadione, MEN), benzene-1,4-diol (hydroquinone, HYQ)), and two non-genotoxic (methyl carbamate (MC) and dimethyl sulfoxide (DMSO)) compounds. A library of S. cerevisiae 4,852 deletion strains, each identifiable by a unique genetic ‘barcode’, were grown in competition; at different time points the ratio between the strains was assessed by quantitative high throughput ‘barcode’ sequencing. The method was validated by comparison to previous genomic phenotyping studies and 90% of the strains identified as MMS-sensitive here were also identified as MMS-sensitive in a much lower throughput solid agar screen. The data provide profiles of proteins and pathways needed for recovery after both genotoxic and non-genotoxic compounds. In addition, a novel role for aromatic amino acids in the recovery after treatment with oxidizing agents was suggested. The role of aromatic acids was further validated; the quinone subgroup of oxidizing agents were extremely toxic in cells where tryptophan biosynthesis was compromised.Unilever (Firm)National Cancer Institute (U.S.) (R01-CA055042 (now R01-ES022872))Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant NIEHS P30-ES002109

Global Transcriptome and Deletome Profiles of Yeast Exposed to Transition Metals

A variety of pathologies are associated with exposure to supraphysiological concentrations of essential metals and to non-essential metals and metalloids. The molecular mechanisms linking metal exposure to human pathologies have not been clearly defined. To address these gaps in our understanding of the molecular biology of transition metals, the genomic effects of exposure to Group IB (copper, silver), IIB (zinc, cadmium, mercury), VIA (chromium), and VB (arsenic) elements on the yeast Saccharomyces cerevisiae were examined. Two comprehensive sets of metal-responsive genomic profiles were generated following exposure to equi-toxic concentrations of metal: one that provides information on the transcriptional changes associated with metal exposure (transcriptome), and a second that provides information on the relationship between the expression of ∼4,700 non-essential genes and sensitivity to metal exposure (deletome). Approximately 22% of the genome was affected by exposure to at least one metal. Principal component and cluster analyses suggest that the chemical properties of the metal are major determinants in defining the expression profile. Furthermore, cells may have developed common or convergent regulatory mechanisms to accommodate metal exposure. The transcriptome and deletome had 22 genes in common, however, comparison between Gene Ontology biological processes for the two gene sets revealed that metal stress adaptation and detoxification categories were commonly enriched. Analysis of the transcriptome and deletome identified several evolutionarily conserved, signal transduction pathways that may be involved in regulating the responses to metal exposure. In this study, we identified genes and cognate signaling pathways that respond to exposure to essential and non-essential metals. In addition, genes that are essential for survival in the presence of these metals were identified. This information will contribute to our understanding of the molecular mechanism by which organisms respond to metal stress, and could lead to an understanding of the connection between environmental stress and signal transduction pathways

A Data Collection Tool to capture a Core Mental Health Dataset within Physical Health Clinical Trials

A Data Collection Tool for a Core Mental Health Dataset in Physical Health Research Studies Project Overview: The overarching aim is to create a seamless and widely acceptable mechanism to collect a common/core set of mental health data (the Core Mental Health Dataset - CMHDS) for all UK physical health study participants so that researchers can analyse the links between physical and mental health. CMHDS Data Collection Tool: The Core Mental Health DataSet (CMHDS) Open Source tool enables the collection of mental health data from participants in physical health studies. The CMHDS data collection tool was extensively co-designed with members of the public through a series of workshops. How participants engage with the tool: From a participant perspective, participants are provided with a URL which provides access to the CMHDS website. The website contains: A Participant Information Sheet A link to a short animation to explain the study in simple terms A consent form with mandatory and optional consents Clear information about how data collected will be used and for which purposes A bespoke mental health history questionnaire Validated questionnaires were included in the original CMHDS roll-out including: GHQ12, PHQ8 and GAD7. However, licenses are required to use some of the validated questionnaires and for this purpose they are not included in the source code here. These questionnaires (and others) can be easily added to a future deployment. Technical details of how to do this are included in the README.txt file. All questionnaires are optional to complete and the questionnaire can be exited at any time. There is an explanation of what each questionnaire entails that participants can read before answering them. The CMHDS website also contains a contact email so that participants can contact the CMHDS team if they have any questions and sources of support for people who have concerns about their mental health. Author Contributions Study Chief Investigator: Professor Kathryn Abel Co-investigators: Dr Pauline Whelan Professor Ann John Professor Paul Dark Professor Nawar Diar Bakerly PPI Lead Contributors Martin Rathfelder Philip Bell Researchers and Focus Group Facilitators Dr Kerry Gutridge Dr Jenni Jardine Auden Edwardes Operational Lead Charlie Stockton-Powdrell Lead Software UX designer - design of the website Simon Foster Software Engineers - development of the web app Henry Gorner Paul Smitton Acknowledgements: This study/project is funded by the NIHR Research for Patient Benefit, Award ID: NIHR201104. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. Thanks to all of our patient and public contributors who attended workshops and helped co-design the CMHDS tool with us

Keeping the spotlight on cytochrome P450

This review describes the recent advances utilizing photosensitizers and visible light to harness the synthetic potential of P450 enzymes. The structures of the photosensitizers investigated to date are first presented along with their photophysical and redox properties. Functional photosensitizers range from organic and inorganic complexes to nanomaterials as well as the biological photosystem I complex. The focus is then on the three distinct approaches that have emerged for the activation of P450 enzymes. The first approach utilizes the in situ generation of reactive oxygen species entering the P450 mechanism via the peroxide shunt pathway. The other two approaches are sustained by electron injections into catalytically competent heme domains either facilitated by redox partners or through direct heme domain reduction. Achievements as well as pitfalls of each approach are briefly summarized. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone