Single cell genomics and characterisation of the atmospheric methane oxidizing clade USC alpha and their response to climate change
Lead Research Organisation:
Heriot-Watt University
Department Name: Sch of Energy, Geosci, Infrast & Society
Abstract
Methane is a very potent greenhouse gas, at least 20 times more effective than carbon dioxide. It has a current atmospheric concentration of about 1.8 ppmv. The largest biogenic sources of atmospheric methane include natural wetlands, rice agriculture, livestock, landfills, termites and oceans. The most important biological sinks for methane in the biosphere are upland soils, especially forest soils which show the greatest methane oxidation consumption capability of any soil ecosystem. Methane from the atmosphere (ie at very low concentrations) is taken up by a specific group of microorganisms in those soils. The bacterial "upland soil cluster alpha" clade (USCa clade) in forest soils, which has been previously detected solely by cultivation-independent molecular biological techniques, is assumed to represent the methanotrophic bacteria adapted to the trace level of atmospheric methane which play an essential part in the removal of methane from the atmosphere. We know from studying the cycles of climatically important gases such as methane that microbial consumption is an extremely important process which is greatly influenced by climate change. Global warming and land use change will have a significant impact on the microorganisms in forest soil by changing the environmental and soil conditions (e.g. increased temperature, changing water content, increased methane and carbon dioxide concentration, deforestation and nitrogen fertilization due to agricultural use).
However, since bacteria of the USCa clade have not be isolated and grown in culture in the laboratory, little is known about the about the identity, the physiology, biochemistry and metabolic capabilities of these microorganisms and how they respond to changes in the environment ie how their activities are regulated in response to environmental change.
Using powerful molecular biology-based techniques, I therefore aim to investigate the identity and abilities of the USCa by sorting single cells of this clade from soil and looking at the DNA of the cells, the genome. Most bacteria and archaea look alike, so we frequently use DNA and RNA sequences to study their roles and activity in nature. I will use the genome information which contains how a microorganism works (the metabolic blueprint of the microorganism) and what it needs in terms of nutrients, to enrich and isolate bacteria of the USCa clade from soils that have high activities in oxidising methane at atmospheric concentrations. The purification of the enzyme responsible for the high-affinity methane oxidation activity from bacteria of the USCa clade will give us significant insight into the regulation of its methane oxidation activity. Additionally, I will apply tools developed by myself and collaborators to analyse total DNA and RNA of soil samples and enrichments, termed metagenomics and metatranscriptomics, respectively. This will further enable us to determine how the USCa bacteria will react to rising temperatures and changes in land use. The use of state-of-the-art techniques will allow me to identify the atmospheric methane oxidizing USCa bacteria in forest soil, and to determine how their activity is controlled and influenced by a changing climate and changes in land use (e.g. deforestation and changes in agricultural practice). In addition, this information will be used to survey for the presence of USCa bacteria in different geographical locations and to look at the long-term effect of different environments on the evolution of these microorganisms.
The results of this project will provide robust mechanistic data with which to predict the impact of climate change and land use on health and functioning of the global sink strength for atmospheric methane in forest/upland soils.
However, since bacteria of the USCa clade have not be isolated and grown in culture in the laboratory, little is known about the about the identity, the physiology, biochemistry and metabolic capabilities of these microorganisms and how they respond to changes in the environment ie how their activities are regulated in response to environmental change.
Using powerful molecular biology-based techniques, I therefore aim to investigate the identity and abilities of the USCa by sorting single cells of this clade from soil and looking at the DNA of the cells, the genome. Most bacteria and archaea look alike, so we frequently use DNA and RNA sequences to study their roles and activity in nature. I will use the genome information which contains how a microorganism works (the metabolic blueprint of the microorganism) and what it needs in terms of nutrients, to enrich and isolate bacteria of the USCa clade from soils that have high activities in oxidising methane at atmospheric concentrations. The purification of the enzyme responsible for the high-affinity methane oxidation activity from bacteria of the USCa clade will give us significant insight into the regulation of its methane oxidation activity. Additionally, I will apply tools developed by myself and collaborators to analyse total DNA and RNA of soil samples and enrichments, termed metagenomics and metatranscriptomics, respectively. This will further enable us to determine how the USCa bacteria will react to rising temperatures and changes in land use. The use of state-of-the-art techniques will allow me to identify the atmospheric methane oxidizing USCa bacteria in forest soil, and to determine how their activity is controlled and influenced by a changing climate and changes in land use (e.g. deforestation and changes in agricultural practice). In addition, this information will be used to survey for the presence of USCa bacteria in different geographical locations and to look at the long-term effect of different environments on the evolution of these microorganisms.
The results of this project will provide robust mechanistic data with which to predict the impact of climate change and land use on health and functioning of the global sink strength for atmospheric methane in forest/upland soils.
Planned Impact
The project will provide essential data on methane cycling in soil, the factors controlling the sink strength of upland soils for methane and how this sink will respond to ongoing climate warming and land use change. It will develop molecular tools to investigate the physiology, activity, distribution and diversity of the key microorganisms (the upland soil cluster alpha, USCa) involved in this process and provide understanding of their response to environmental changes.
This research will have high impact because of the timely and urgent need of science and society, to better understand and predict the impacts of global climate change on environments worldwide. In particular because the uptake of methane by soils represents the largest and most effective biological sink for atmopsheric methane.
This research should benefit scientists worlwide, involved in research on terrestrial microbiology and global climate change, and will be of great interest to stakeholders involved in the environment and climate change (Defra, DECC, the MetOffice, IPCC, the Environment Agency). I will participate in end-user led meetings during the course of the project and engage with key individuals in the scientific community who are interested in integrating the data from this project in global flux models of methane and predictions concerning global climate change (Le Quere and Suntharalingum at UEA and Smith at Aberdeen). Discussions will als0 be initiated with Forest Research to advise government on important outcomes related to greenhouse gas emissions from natural forests and biomass plantations.
As outlined in more detail in the Pathways to Impact, there are other potential applications of this work. The possibility of exploiting new, high-affinity methane oxidation enzyme systems and methanotrophs during this reserach could also benefit biotechnology and industries. I will make contact with industries already exploiting methanotrophs in biotechnology and companies exploring new ways of removing low concentrations of methane from waste gases, e.g. in coal mines (UK Mining Industry). This would be followed up through meetings with the host institution's respective research and enterprise offices, which have expertise in biotechnology patents.
The results of this topical and globally relevant research involving: identification of microbial key players in the uptake of atmopsheric methane in upland soils, characterization of their physiology and nutritional requirements and investigation of the environmental factors controlling their global activity and distribution and their response to climate change and land use, will capture the public's attention. Therefore an attractive and informative web site is proposed, including e.g. video footage from field work and from work in the lab. The web site will be used to inform the public about the field of microbiology, methane and other atmospheric trace gases, and the connections to climate change, in an entertaining and easy to grasp way. School visits throughout the duration of the project are also proposed, as well as demonstrations in and outside the laboratory, to engage pupils and teachers in science, especially of environmental microbiology. Popular articles written for Microbiology Today, The Microbiologist, The New Scientist and Planet Earth will further publicise this work to the wider community.
The Applicant, together with the multidisciplinary collaborative team, has an excellent record of engaging with the public, e.g. via school visits and the popular scientific press and radio.
This approach will help to make the public more aware about the impacts of climate change and the role of microorganisms involved in biogeochemical cycles, and sensitize them to the environmental issues arising from global warming.
This research will have high impact because of the timely and urgent need of science and society, to better understand and predict the impacts of global climate change on environments worldwide. In particular because the uptake of methane by soils represents the largest and most effective biological sink for atmopsheric methane.
This research should benefit scientists worlwide, involved in research on terrestrial microbiology and global climate change, and will be of great interest to stakeholders involved in the environment and climate change (Defra, DECC, the MetOffice, IPCC, the Environment Agency). I will participate in end-user led meetings during the course of the project and engage with key individuals in the scientific community who are interested in integrating the data from this project in global flux models of methane and predictions concerning global climate change (Le Quere and Suntharalingum at UEA and Smith at Aberdeen). Discussions will als0 be initiated with Forest Research to advise government on important outcomes related to greenhouse gas emissions from natural forests and biomass plantations.
As outlined in more detail in the Pathways to Impact, there are other potential applications of this work. The possibility of exploiting new, high-affinity methane oxidation enzyme systems and methanotrophs during this reserach could also benefit biotechnology and industries. I will make contact with industries already exploiting methanotrophs in biotechnology and companies exploring new ways of removing low concentrations of methane from waste gases, e.g. in coal mines (UK Mining Industry). This would be followed up through meetings with the host institution's respective research and enterprise offices, which have expertise in biotechnology patents.
The results of this topical and globally relevant research involving: identification of microbial key players in the uptake of atmopsheric methane in upland soils, characterization of their physiology and nutritional requirements and investigation of the environmental factors controlling their global activity and distribution and their response to climate change and land use, will capture the public's attention. Therefore an attractive and informative web site is proposed, including e.g. video footage from field work and from work in the lab. The web site will be used to inform the public about the field of microbiology, methane and other atmospheric trace gases, and the connections to climate change, in an entertaining and easy to grasp way. School visits throughout the duration of the project are also proposed, as well as demonstrations in and outside the laboratory, to engage pupils and teachers in science, especially of environmental microbiology. Popular articles written for Microbiology Today, The Microbiologist, The New Scientist and Planet Earth will further publicise this work to the wider community.
The Applicant, together with the multidisciplinary collaborative team, has an excellent record of engaging with the public, e.g. via school visits and the popular scientific press and radio.
This approach will help to make the public more aware about the impacts of climate change and the role of microorganisms involved in biogeochemical cycles, and sensitize them to the environmental issues arising from global warming.
People |
ORCID iD |
Jennifer Pratscher (Principal Investigator / Fellow) |
Publications
Ilieva V
(2021)
Assembly of Bacterial Genome Sequences from Metagenomes of Spacecraft Assembly Cleanrooms.
in Microbiology resource announcements
Wagstaff BA
(2021)
Assessing the Toxicity and Mitigating the Impact of Harmful Prymnesium Blooms in Eutrophic Waters of the Norfolk Broads.
in Environmental science & technology
Guillen Ferrari D
(2019)
Assessment of the use of compost stability as an indicator of alkane and aromatic hydrocarbon degrader abundance in green waste composting materials and finished composts for soil bioremediation application.
in Waste management (New York, N.Y.)
Williams BT
(2019)
Bacteria are important dimethylsulfoniopropionate producers in coastal sediments.
in Nature microbiology
Yang Y
(2022)
DNA-SIP reveals an overlooked methanotroph, Crenothrix sp., involved in methane consumption in shallow lake sediments.
in The Science of the total environment
Wang J
(2018)
Draft Genome Sequence of Methylocella silvestris TVC, a Facultative Methanotroph Isolated from Permafrost.
in Genome announcements
Macey MC
(2018)
Draft Genome Sequences of Obligate Methylotrophs Methylovorus sp. Strain MM2 and Methylobacillus sp. Strain MM3, Isolated from Grassland Soil.
in Microbiology resource announcements
Pratscher J
(2021)
Extraction of Microbial Cells from Environmental Samples for FISH Approaches.
in Methods in molecular biology (Clifton, N.J.)
Macey MC
(2020)
Impact of plants on the diversity and activity of methylotrophs in soil.
in Microbiome
Wagstaff BA
(2018)
Insights into toxic Prymnesium parvum blooms: the role of sugars and algal viruses.
in Biochemical Society transactions
Carrión O
(2019)
Methanethiol and Dimethylsulfide Cycling in Stiffkey Saltmarsh.
in Frontiers in microbiology
Larke-Mejía NL
(2019)
Novel Isoprene-Degrading Proteobacteria From Soil and Leaves Identified by Cultivation and Metagenomics Analysis of Stable Isotope Probing Experiments.
in Frontiers in microbiology
Crombie A
(2018)
Poplar phyllosphere harbors disparate isoprene-degrading bacteria
in Proceedings of the National Academy of Sciences
Ritson JP
(2021)
Towards a microbial process-based understanding of the resilience of peatland ecosystem service provisioning - A research agenda.
in The Science of the total environment
Pratscher J
(2018)
Unravelling the Identity, Metabolic Potential and Global Biogeography of the Atmospheric Methane-Oxidizing Upland Soil Cluster a.
in Environmental microbiology
Description | So far we have discovered that the so-far unidentified bacteria in soil that can take up the greenhouse gas methane at atmospheric concentrations, probably belong to the family Beijerinckiaceae. Parts of their genome indicate that they can potentially use a wide range of carbon sources and they further seem to possess one or several mechanisms for copper-tolerance. We now have the complete 16S rRNA gene sequence for species of this upland soil cluster, which enabled us for the first time to conduct a global environmental survey of this cluster in existing databases, showing that these upland soil bacteria are present in environments we previously had not anticipated (e.g. subsurface and underground, volcanic and high altitude environments). This significantly expands the range of environments these bacteria were thought to be present, and opens new questions about their environmental role (e.g. in biofilms) and activity. We are currently also in the process of applying this knowledge on potential methane biofilter applications. |
Exploitation Route | Findings on the regulation of the activity of atmospheric methane oxidising bacteria in soils might influence de- and reforestation procedures and might be of use for industry in the form of methane biofilters. |
Sectors | Environment,Manufacturing, including Industrial Biotechology |
URL | http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.14036/full |
Description | Participation in workshops on "Towards a microbial process-based understanding of the resilience of UK peatland systems", funded by a NERC grant from the UK Climate Resilience Programme (2019/2020, organised by University of Manchester). These workshops have already resulted in one policy brief (see below). |
Geographic Reach | National |
Policy Influence Type | Participation in a guidance/advisory committee |
URL | https://peatlandmicrobes.com/policybrief/ |
Description | Chinese Government Scholarship |
Amount | £14,000 (GBP) |
Funding ID | 201706010303 |
Organisation | Chinese Scholarship Council |
Sector | Charity/Non Profit |
Country | China |
Start | 02/2018 |
End | 10/2018 |
Description | Methane oxidising bacteria in biofilm environments |
Amount | £100,000 (GBP) |
Funding ID | James Watt scholarship |
Organisation | Heriot-Watt University |
Sector | Academic/University |
Country | United Kingdom |
Start | 06/2019 |
End | 12/2022 |
Title | Methylocella silvestris TVC draft genome |
Description | Draft genome sequence of Methylocella silvestris TVC, a facultative methanotrophic bacterium isolated from Permafrost |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | This is the first draft genome of a novel isolated facultative methanotrophic Methylocella bacterium isolated from Permafrost, providing insight into the potential metabolism and climate response of this versatile microbial group. |
URL | https://www.ncbi.nlm.nih.gov/nuccore/PDZR00000000 |
Title | USCa draft genome |
Description | First draft genome of an atmospheric methane oxidising bacterium of the USCalpha clade. |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | This is the first draft genome of an atmospheric methane oxidising bacterium of the USCalpha clade, providing insight into the metabolism and regulation of this crucial group. |
URL | https://www.ncbi.nlm.nih.gov/nuccore/PEFW00000000 |
Description | Permafrost |
Organisation | University of East Anglia |
Department | School of Environmental Sciences UEA |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Heriot-Watt's (HW) part on this collaboration is to analyse the microbial diversity and activity in permafrost soil samples, and to identify the active methanogenic and methanotrophic microorganisms involved in methane cycling in these environments. |
Collaborator Contribution | The HW results are added to fungal diversity analyses (Manchester) and flux-measurements (University of Stirling, University of Edinburgh) and all will be integrated into process-based biogeochemical and biophysical modelling. Heriot-Watt also provides field sampling and soil samples. |
Impact | This collaboration already resulted in the isolation of a new methanotrophic bacterial strain from permafrost soil: Wang, J., Geng, K., Ul Haque, M.F., Crombie, A., Street, L., Wookey, P., Ma, K., Murrell, J.C., Pratscher, J. (2018). "Draft genome sequence of Methylocella silvestris TVC, a facultative methanotroph isolated from permafrost". Genome Announcements DOI: 10.1128/genomeA.00040-18. We are currently preparing another publication on our research results. This collaboration also lead to the invitation and contribution to series workshop on "Towards a microbial process-based understanding of the resilience of UK peatland systems", funded by a NERC grant from the UK Climate Resilience Programme (2019/2020, organised by University of Manchester). These workshops have already resulted in one policy brief (https://peatlandmicrobes.com/policybrief/), 1 published review, and 1 more article submission. |
Start Year | 2014 |
Description | Permafrost |
Organisation | University of Manchester |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Heriot-Watt's (HW) part on this collaboration is to analyse the microbial diversity and activity in permafrost soil samples, and to identify the active methanogenic and methanotrophic microorganisms involved in methane cycling in these environments. |
Collaborator Contribution | The HW results are added to fungal diversity analyses (Manchester) and flux-measurements (University of Stirling, University of Edinburgh) and all will be integrated into process-based biogeochemical and biophysical modelling. Heriot-Watt also provides field sampling and soil samples. |
Impact | This collaboration already resulted in the isolation of a new methanotrophic bacterial strain from permafrost soil: Wang, J., Geng, K., Ul Haque, M.F., Crombie, A., Street, L., Wookey, P., Ma, K., Murrell, J.C., Pratscher, J. (2018). "Draft genome sequence of Methylocella silvestris TVC, a facultative methanotroph isolated from permafrost". Genome Announcements DOI: 10.1128/genomeA.00040-18. We are currently preparing another publication on our research results. This collaboration also lead to the invitation and contribution to series workshop on "Towards a microbial process-based understanding of the resilience of UK peatland systems", funded by a NERC grant from the UK Climate Resilience Programme (2019/2020, organised by University of Manchester). These workshops have already resulted in one policy brief (https://peatlandmicrobes.com/policybrief/), 1 published review, and 1 more article submission. |
Start Year | 2014 |
Description | Permafrost |
Organisation | University of Stirling |
Department | School of Natural Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Heriot-Watt's (HW) part on this collaboration is to analyse the microbial diversity and activity in permafrost soil samples, and to identify the active methanogenic and methanotrophic microorganisms involved in methane cycling in these environments. |
Collaborator Contribution | The HW results are added to fungal diversity analyses (Manchester) and flux-measurements (University of Stirling, University of Edinburgh) and all will be integrated into process-based biogeochemical and biophysical modelling. Heriot-Watt also provides field sampling and soil samples. |
Impact | This collaboration already resulted in the isolation of a new methanotrophic bacterial strain from permafrost soil: Wang, J., Geng, K., Ul Haque, M.F., Crombie, A., Street, L., Wookey, P., Ma, K., Murrell, J.C., Pratscher, J. (2018). "Draft genome sequence of Methylocella silvestris TVC, a facultative methanotroph isolated from permafrost". Genome Announcements DOI: 10.1128/genomeA.00040-18. We are currently preparing another publication on our research results. This collaboration also lead to the invitation and contribution to series workshop on "Towards a microbial process-based understanding of the resilience of UK peatland systems", funded by a NERC grant from the UK Climate Resilience Programme (2019/2020, organised by University of Manchester). These workshops have already resulted in one policy brief (https://peatlandmicrobes.com/policybrief/), 1 published review, and 1 more article submission. |
Start Year | 2014 |
Description | Permafrost |
Organisation | Wellcome Trust |
Department | Wellcome Trust Centre for Cell Biology |
Country | United Kingdom |
Sector | Charity/Non Profit |
PI Contribution | Heriot-Watt's (HW) part on this collaboration is to analyse the microbial diversity and activity in permafrost soil samples, and to identify the active methanogenic and methanotrophic microorganisms involved in methane cycling in these environments. |
Collaborator Contribution | The HW results are added to fungal diversity analyses (Manchester) and flux-measurements (University of Stirling, University of Edinburgh) and all will be integrated into process-based biogeochemical and biophysical modelling. Heriot-Watt also provides field sampling and soil samples. |
Impact | This collaboration already resulted in the isolation of a new methanotrophic bacterial strain from permafrost soil: Wang, J., Geng, K., Ul Haque, M.F., Crombie, A., Street, L., Wookey, P., Ma, K., Murrell, J.C., Pratscher, J. (2018). "Draft genome sequence of Methylocella silvestris TVC, a facultative methanotroph isolated from permafrost". Genome Announcements DOI: 10.1128/genomeA.00040-18. We are currently preparing another publication on our research results. This collaboration also lead to the invitation and contribution to series workshop on "Towards a microbial process-based understanding of the resilience of UK peatland systems", funded by a NERC grant from the UK Climate Resilience Programme (2019/2020, organised by University of Manchester). These workshops have already resulted in one policy brief (https://peatlandmicrobes.com/policybrief/), 1 published review, and 1 more article submission. |
Start Year | 2014 |
Description | "Microbial Data Analysis Workshop on the MRC Cloud infrastructure for Microbial Bioinformatics (Climb)", 14-15/05/19, Heriot-Watt University, Edinburgh, UK |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | We organised the first CLIMB (Cloud Infrastructure for Microbial Bioinformatics) workshop in Scotland. 50 PIs, Postdocs, and PhD student attended the workshop. |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.climb.ac.uk/microbial-data-analysis-workshop/ |
Description | EuC1 Meeting - Microbial C1 Metabolism (Nijmegen, NL, 17-18 May 2018) |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | EuC1 Meeting - Microbial C1 Metabolism (Nijmegen, NL, 17-18 May 2018). The objective of this European meeting is to foster multidisciplinary collaboration focusing on Microbial C1 metabolism mechanism. The project focuses on to the establishment of a network-wide collaborative research activity to gain fundamental understanding and knowledge of microbial C1 metabolism, and to identify opportunities for future multidisciplinary projects. |
Year(s) Of Engagement Activity | 2018 |
Description | Invited talk on "Single Cell Genomics" to Environmental Genomics and Advanced Microbiological Techniques workshop, April 15 2019, UEA, Norwich, UK |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | 40 PhD students and Postdocs attended the workshop, which sparked a lot of creative and productive dicussion and provided them with opportunities to implement these techniques and approaches into their current and future projects. |
Year(s) Of Engagement Activity | 2019 |
Description | Invited talk to workshop "Methane Economy" at Queen's University Belfast (January 2018) |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Invited talk to workshop "Methane Economy" at Queen's University Belfast (January 2018). Title of talk: Illuminating the soil methane sink. |
Year(s) Of Engagement Activity | 2018 |
Description | Lectures for the undergraduate module "Microbial Cell Biology" at the School of Biological Sciences, UEA. (January 2017 and January 2018) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Undergraduate students |
Results and Impact | Lectures on "Metagenomics and its uses" and "Bioinformatics analysis of high throughput sequence data" for the undergraduate module "Microbial Cell Biology" at the School of Biological Sciences, University of East Anglia (January 2017 and January 2018). |
Year(s) Of Engagement Activity | 2017,2018 |
Description | NERC-funded workshop on "Towards a microbial process-based understanding of the resilience of UK peatland systems" |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Invited and contribution to series workshop on "Towards a microbial process-based understanding of the resilience of UK peatland systems", funded by a NERC grant from the UK Climate Resilience Programme (2019/2020, organised by University of Manchester). These workshops have already resulted in one policy brief (https://peatlandmicrobes.com/policybrief/), 1 published review, and 1 more article submission. |
Year(s) Of Engagement Activity | 2019 |
URL | https://peatlandmicrobes.com/policybrief/ |