Markus Andrews

Test manufactureThe DNA plasmid encoding the fluoride biosensor used in this study was assembled using Gibson assembly (New England Biolabs, Cat#E2611S) and purified using a Qiagen QIAfilter Midiprep Kit (QIAGEN, Cat#12143). Its coding sequence consists of the crcB fluoride riboswitch from Bacillus cereus regulating the production of the enzyme catechol 2,3-dioxygenase, all expressed under the constitutive E. coli sigma 70 consensus promoter J2311939. A complete sequence of the plasmid used is available on Addgene with accession number 128810 (pJBL7025) [https://www.addgene.org/128810/].Cell-free biosensing reactions used in the tests were set up according to previously established protocols20,40. Briefly, reactions consist of cleared cellular extract, a reagent mix containing amino acids, buffering salts, crowding agents, enzymatic substrate, and an energy source, and a reaction-specific mix of template DNA and sodium fluoride in an approximately 30/30/40 ratio (Supplementary Table 3). Test reactions contained no sodium fluoride, while positive control reactions were supplemented with 1 mM sodium fluoride to induce gene expression. Template DNA concentration for both sets of reactions was 5 nM, determined by the maximal template concentration at which no color change was observed in the absence of fluoride.During reaction setup, master mixes of cellular extract, reagent mix, and template mix were prepared for both test and positive control reactions in 1.7 mL microcentrifuge tubes. Individual reactions were then aliquoted into 20 µL volumes in PCR tube strips for lyophilization. After aliquoting on ice, PCR tube caps were pierced with a pin, strips were wrapped in aluminum foil, then the wrapped strips were immersed in liquid nitrogen for freeze-drying for approximately 3 min. Reactions were immediately transferred to a Labconco FreeZone 2.5 Liter −84 °C Benchtop Freeze-Dryer (Cat# 710201000) with a condenser temperature of −84 °C and pressure of 0.04 mbar and freeze-dried overnight (≥16 h).After freeze-drying, tests were vacuum sealed (KOIOS Vacuum Sealer Machine, Amazon, Amazon Standard Identification Number (ASIN) B07FM3J6JF) in a food saver bag (KOIS Vacuum Sealer Bag, Amazon, ASIN B075KKWFYN), along with a desiccant (Dri-Card Desiccants, Uline, Cat# S-19582) (Supplementary Fig. 3). Vacuum sealed reactions were then paced in a light-protective outer bag (Mylar open-ended food bags, Uline, Cat# S-11661) and impulse heat-sealed (Metronic 8-inch Impulse Bag Sealer, Amazon, ASIN B06XC76JVZ) before shipping. Tests were also shipped with single-use 20 µL micropipettes (MICROSAFE® 20 µL, Safe-Tec LLC, Cat# 1020) for field operation.Test-kit shipment to Nakuru County, KenyaA first shipment of biosensor tests was used to assess 33 water samples from the first 16 households surveyed. All of these tests resulted in a faint yellow color, regardless of water source or fluoride concentration established via fluorimeter. This was likely caused by thermal degradation of the tests during shipment with the commercial shipping agency. While previous studies report shelf stability for up to a year20,41, these figures were derived from storage in temperature-controlled laboratory conditions. Commercial shipment routes from Illinois, USA to Nairobi, Kenya pass through extremely hot regions, e.g., Dubai for this particular shipment. These conditions were much different from those in the previous study usability study in Costa Rica in which tests were transported by commercial air, with gentler shipping and storage conditions20. A laboratory investigation of test temperature stability indicated that elevated storage temperatures can indeed cause test components to degrade, resulting in a faint yellow color upon rehydration consistent with field observations (Supplementary Fig. 2).The next batch of tests was therefore shipped refrigerated on January 25th, 2022, which we hypothesized would extend the tests’ shelf stability to align with earlier findings. After the tests were made and packaged, they were placed in a polystyrene foam-lined container before being covered with a NanoCool refrigeration system (Peli BioThermal). The container was then sealed shut and shipped using a standard commercial shipping service. This batch of tests was held in customs, refrigerated, until release on February 28th, 2022. These tests were used in the field from March 5th to March 14th, 2022 to generate the data on test accuracy reported in this manuscript.As discoloration due to thermal degradation could confound the intended yellow hue in the presence of fluoride (i.e., false positives), we assessed test accuracy using only tests that had been refrigerated during shipping and transport to participants’ houses. The 33 water samples from the first 16 households were therefore excluded from analysis of test accuracy.Participant recruitmentParticipants were recruited from six sublocations (Kelelwet, Kipsimbol, Kigonor, Parkview, Lalwet, and Mwariki) in Barut Ward within Nakuru County (Supplementary Fig. 4, geographic information adapted from OpenStreetMap42). This location was chosen because of high fluoride levels and familiarity with the communities by the study team.Before any data were collected, community meetings were held in each sub-location to discuss study goals and objectives. After obtaining permission from the community and village assistant chiefs to conduct research, local community mobilizers were engaged to assist with identifying households eligible for participation. Individuals who were 18 years or older, had lived in Nakuru country for more than three months, relied on local water sources for drinking, had a child in the household, were willing to discuss their household water situation, and provide a sample of each source of water in the household for fluoride testing were eligible. We sought to recruit 10–12 participants from each of the five sublocations to ensure a range of sociodemographic characteristics and drinking water sources. Having a child resident was a criterion in order to elucidate community understandings about fluorosis in children.Data collectionAfter obtaining informed written consent, participants participated in a 30-min survey (cf. Supplementary Fig. 1 for a graphical overview of data collection). Topics included household sociodemographic information, knowledge, attitudes, and behaviors about fluoride and fluorosis, and household water insecurity using the validated Household Water Insecurity Experiences (HWISE) scale43. The 12 HWISE items query the frequency of experiences with water insecurity in the prior month; “never” is scored 0, “often/always” scored 3, for a range of 0–36. These data were collected to be able to investigate if user experiences or attitudes about testing varied by experiences with fluorosis or water insecurity. Participants were also asked about the number of sources of their water and willingness to provide and test water samples. Survey responses were recorded on tablets using Open Data Kit (ODK)44.After completion of the survey, participants provided 1–3 samples of water from different household sources. They then received a brief (~5 min) explanation of the testing process, and then tested their own household samples using the fluoride biosensor tests. Each test consisted of a microtube that was a positive control, and a second microtube in which the sample of interest was tested. To test their samples, participants first removed the tests from the light-protective foil pouch and vacuum sealed pouch containing desiccant, both of which were then discarded (Supplementary Fig. 3). A micropipette was then filled with 20 µL water by slowly immersing it to the fill line. To dispense the water, the thumb and index finger were used to cover the holes in the micropipette while the bulb was squeezed with the other hand. The reactions were then incubated at ambient temperature for up to six hours, shorter if there was a visible color change. During this incubation time, participants were asked to check hourly for yellow color change and note the time taken for it to occur. Tests were expected to turn yellow if fluoride levels were ≥1.5 ppm, with no color change for tests of water below this level. All positive controls were expected to turn yellow. Color change was read after placing reactions against a white background for visual contrast.The study team returned to conduct a second survey on user experiences with the testing process and to test the water samples using the gold-standard photometer within 6 h. Participants were asked about their experiences with the testing procedure as well as their interpretation of the color of the results of the sample and control tests. Photographs of the completed reactions were also taken at this time. Finally, quantitative fluoride measurements were taken by the field team with a Hanna Instruments Fluoride High Range Photometer Kit (Cat# HI97739C), a gold-standard method used to assess the accuracy of the bioengineered tests. Photometry results on actual measured fluoride concentrations of water samples were shared with and explained to participants. At the conclusion of the second survey, each participant was given KES 500 (USD 4.30) as remuneration for the time and effort spent participating in the research. Each participating household was also given a ceramic drinking water filter.Data were collected from November 16th to November 23rd, 2021 and March 5th to March 14th, 2022. During surveying and water testing, participants and research assistants maintained COVID-19 protocols as per the local area guidelines. Study staff were vaccinated, maintained appropriate social distancing, sanitized hands, and cleaned field tools after each household visit.Data analysisData were exported from ODK into Microsoft Excel for analysis. Basic descriptive statistics were performed to describe participant socio-demographics and experiences with usability, including if participants’ interpretation of color change matched that of study staff. Open-ended items about fluoride and fluorosis knowledge, attitudes, and behavior were grouped thematically and coded independently by two authors. Knowledge-related responses were characterized as “correct” if consistent with conventional biomedical understanding, “incorrect”, or unfamiliar.Tests were classified as ‘ON’ by the Kenya-based field team if they were visibly yellow after six hours, and ‘OFF’ if there was no observable color change by eye. These assessments were independently validated by the US-based team from photographs of the completed tests. Tests classified as ‘ON’ were marked true positive if they corresponded to a photometer measured fluoride concentration ≥1.5 ppm, and false positive if they corresponded to a photometer measured fluoride concentration More

Land area for afforestation with RE-based SWRO desalinationRestoration land37 and bare land areas22 with the following water stress conditions were determined to be areas where forests could grow if irrigated with a secure water supply. The projected water stress, water supply and demand data for the decade 2040 are used. The renewable water resources in these areas were not considered sufficient to sustain forest growth.

Land nodes that lie in high (40%  More

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Communities in Gansu province in China store snow in the winter months for use in the dry summers.Credit: Hai Ying/EPA/Shutterstock

Among the world’s ‘poly-crises’, the crisis of water is one of the most urgent. Worldwide, around 2 billion people lacked access to safe drinking water in 2020; and an estimated 1.7 billion did not have even basic sanitation. Every year, more than 800,000 people die from diarrhoea, because of unsafe drinking water and a lack of sanitation. Most of those are in low- and lower-middle income countries. This is a mind-boggling and unacceptable situation. Even more so in an age when huge investments are being made in instant video calling, personalized medicine and talk of inhabiting other planets.In 2015, the international community declared tackling the water crisis one of the United Nations Sustainable Development Goals (SDGs). The sixth SDG commits the world to “ensure availability and sustainable management of water and sanitation for all”. But the UN acknowledges that SDG 6 is “alarmingly off track”.International diplomacy is finally starting to get its act together. In March, world leaders will assemble in New York City for the UN 2023 Water Conference. It will be the first such event in nearly half a century, a fact that by itself should shame us all.Last October, the UN published the results of a consultation with government representatives as well as specialist and stakeholder communities on their priorities for the conference. Around 12% of respondents were from education, science and technology fields. The consensus was that data and evidence, improved access to knowledge (including Indigenous and local knowledge) and open research will be essential to getting SDG 6 back on track. Delegates attending the March conference will be looking to harness the full spectrum of established water sources and technologies, including freshwater and rainwater sources, treated groundwater, desalinated seawater and hydropower.There’s a wealth of knowledge already out there, in the form of established technologies, innovative alternatives and research that captures centuries-old knowledge and the practices of communities themselves. In the past, such knowledge has been ignored, or what has been learnt has been forgotten. Twenty years ago, for example, the UN invested in a major piece of research that captured examples of how communities living in water-stressed regions have used research and innovation to access water. The research highlighted, for example, how people in arid regions of China store snow in cellars during the winter that can then be melted for use in the summer months.Prerequisites for tackling the water crisis include consolidating what is already known and building on that knowledge. That’s why on 19 January, the Nature Portfolio of journals launched Nature Water. This journal will provide a space for all researchers — including those in natural and social sciences, and in engineering — to collectively contribute their knowledge, insights and the results of their learning, so that the world is on a more equitable and sustainable track. The launch issue includes research in fundamental, applied and social science, as well as opinion and analysis. Our editorial teams are committed to facilitating open science1.Some paths forward are clear. Damir Brdjanovic at the IHE Delft Institute for Water Education in the Netherlands writes in Nature Water that there’s a vast body of research on alternatives to sewered sanitation — and how to use less or no water to safely dispose of faecal matter and inactivate pathogens2. There are alternatives to the flush toilet and underground, piped sewer networks. And Rongrong Xu at the Southern University of Science and Technology in Shenzhen, China, and colleagues report that there are ways to create hydropower, especially in Africa and Asia, without the same environmental and social impacts3.However, research does not exist in a vacuum. The representatives of low- and middle-income countries also want to prioritize funding. The South African government, in its response to the UN consultation, says that the annual cost to meet the SDG water and sanitation targets is between 2.3% and 2.7% of the country’s gross domestic product (between US$7 billion and $7.6 billion annually). A project called the Global Commission on the Economics of Water, co-chaired by economist Mariana Mazzucato and climate scientist Johan Rockström (among others), is promising “new thinking on economics and governance” in time for the conference.Conflict theoryThose who will be attending the conference in March also told the UN they want to see international cooperation be made a priority for water and sanitation, especially in an era of heightened geopolitical tensions. More than 25 years ago, former vice-president of the World Bank Ismail Serageldin famously wrote that twenty-first-century conflicts would be over water, rather than oil. We are fortunate that this has not yet happened, although Serageldin told Nature that relations between countries that share water sources are worsening. Egypt is formally in dispute with Ethiopia over dam-building projects on the Nile River; the same is true of India and Pakistan in the Indus River Basin.In its response to the UN, Egypt’s delegation observed that the majority of people rely on water sources that are shared between nations, most of which lack formal agreements, including all-important data sharing agreements. Rhea Verbeke, at the Catholic University of Leuven in Belgium, writes in Nature Water of the “sobering experience” of seeing no external submissions to an open database on water purification that was created more than one year ago4.The delegates assembling in New York need to accept that their countries’ visions will not be realized until all nations can somehow carve out a path to cooperate amid tension and conflict. Research can help to provide at least some of the right language, which is why it needs to be taken on board when decisions are being made. We in the Nature Portfolio intend to play our fullest part to make that happen. More